Transient protection of normal cells during chemotherapy

ABSTRACT

This invention is in the area of improved compounds, compositions and methods of transiently protecting healthy cells, and in particular hematopoietic stem and progenitor cells (HSPC) as well as renal cells, from damage associated with DNA damaging chemotherapeutic agents. In one aspect, improved protection of healthy cells is disclosed using disclosed compounds that act as highly selective and short, transiently-acting cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors when administered to subjects undergoing DNA damaging chemotherapeutic regimens for the treatment of proliferative disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/222,873, filed Apr. 5, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/112,360, filed Aug. 24, 2018, now U.S. Pat. No.10,966,984, issued, Apr. 6, 2021, which is a continuation of U.S. patentapplication Ser. No. 15/342,990, filed Nov. 3, 2016, now U.S. Pat. No.10,085,992, issued Oct. 2, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/212,430, filed Mar. 14, 2014, now U.S. Pat. No.9,487,530, issued Nov. 8, 2016, which claims the benefit of U.S.Provisional Patent Application No. 61/798,772, filed Mar. 15, 2013, U.S.Provisional Patent Application No. 61/861,374, filed on Aug. 1, 2013,U.S. Provisional Patent Application No. 61/911,354, filed on Dec. 3,2013, and U.S. Provisional Patent Application No. 61/949,786, filed onMar. 7, 2014. The entirety of each of these applications is herebyincorporated by reference for all purposes.

GOVERNMENT INTEREST

This invention was made with government support under Grant No.5R44AI084284 awarded by the National Institutes of Allergy andInfectious Disease. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention is in the area of improved compounds, compositions andmethods of transiently protecting healthy cells, and in particularhematopoietic stem and progenitor cells (HSPC) as well as renal cells,from damage associated with DNA damaging chemotherapeutic agents. In oneaspect, improved protection of healthy cells is disclosed usingdisclosed compounds that act as highly selective and short,transiently-acting cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors whenadministered to subjects undergoing DNA damaging chemotherapeuticregimens for the treatment of proliferative disorders.

BACKGROUND

Chemotherapy refers to the use of cytotoxic (typically DNA damaging)drugs to treat a range of proliferative disorders, including cancer,tumors, psoriasis, arthritis, lupus and multiple sclerosis, amongothers. Chemotherapeutic compounds tend to be non-specific and,particularly at high doses, toxic to normal, rapidly dividing cells.This often leads to a variety of side effects in patients undergoingchemotherapy.

Bone marrow suppression, a severe reduction of blood cell production inbone marrow, is one such side effect. It is characterized by bothmyelosuppression (anemia, neutropenia, agranulocytosis, andthrombocytopenia) and lymphopenia. Neutropenia is characterized by aselective decrease in the number of circulating neutrophils and anenhanced susceptibility to bacterial infections. Anemia, a reduction inthe number of red blood cells or erythrocytes, the quantity ofhemoglobin, or the volume of packed red blood cells (characterized by adetermination of the hematocrit) affects approximately 67% of cancerpatients undergoing chemotherapy in the United States. See BioWorldToday, page 4, Jul. 23, 2002. Thrombocytopenia is a reduction inplatelet number with increased susceptibility to bleeding. Lymphopeniais a common side-effect of chemotherapy characterized by a reduction inthe number of circulating lymphocytes (also called T- and B-cells).Lymphopenic patients are predisposed to a number of types of infections.

Myelosuppression continues to represent the major dose-limiting toxicityof cancer chemotherapy, resulting in considerable morbidity along withthe potential need to require a reduction in chemotherapy doseintensity, which may compromise disease control and survival.Considerable evidence from prospective and retrospective randomizedclinical trials clearly shows that chemotherapy-induced myelosuppressioncompromises long-term disease control and survival (Lyman, G. H.,Chemotherapy dose intensity and quality cancer care (Oncology (WillistonPark), 2006. 20(14 Suppl 9): p. 16-25)). Furthermore, treatment regimensfor, for example, lung, breast, and colorectal cancer recommended in theNational Comprehensive Cancer Network guidelines are increasinglyassociated with significant myelosuppression yet are increasinglyrecommended for treating early-stage disease as well as advanced-stageor metastatic disease (Smith, R. E., Trends in recommendations formyelosuppressive chemotherapy for the treatment of solid tumors. J NatlCompr Canc Netw, 2006.4(7): p. 649-58). This trend toward more intensivetreatment of patients with cancer creates demand for improved measuresto minimize the risk of myelosuppression and complications whileoptimizing the relative dose-intensity.

In addition to bone marrow suppression, chemotherapeutic agents canadversely affect other healthy cells such as renal epithelial cells,resulting potentially in the development of acute kidney injury due tothe death of the tubular epithelia. Acute kidney injury can lead tochronic kidney disease, multi-organ failure, sepsis, and death.

One mechanism to minimize myelosuppression, nephrotoxicity, and otherchemotherapeutic cytotoxicities is to reduce the planned dose intensityof chemotherapies. Dose reductions or cycle delays, however, diminishthe effectiveness and ultimately compromise long-term disease controland survival.

Small molecules have been used to reduce some of the side effects ofcertain chemotherapeutic compounds. For example, leukovorin has beenused to mitigate the effects of methotrexate on bone marrow cells and ongastrointestinal mucosa cells. Amifostine has been used to reduce theincidence of neutropenia-related fever and mucositis in patientsreceiving alkylating or platinum-containing chemotherapeutics. Also,dexrazoxane has been used to provide cardioprotection from anthracyclineanti-cancer compounds. Unfortunately, there is concern that manychemoprotectants, such as dexrazoxane and amifostine, can decrease theefficacy of chemotherapy given concomitantly.

Additional chemoprotectant therapies, particularly with chemotherapyassociated anemia and neutropenia, include the use of growth factors.Hematopoietic growth factors are available on the market as recombinantproteins. These proteins include granulocyte colony stimulating factor(G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF)and their derivatives for the treatment of neutropenia, anderythropoietin (EPO) and its derivatives for the treatment of anemia.However, these recombinant proteins are expensive. Moreover, EPO hassignificant toxicity in cancer patients, leading to increasedthrombosis, relapse and death in several large randomized trials. G-CSFand GM-CSF may increase the late (>2 years post-therapy) risk ofsecondary bone marrow disorders such as leukemia and myelodysplasia.Consequently, their use is restricted and not readily available any moreto all patients in need. Further, while growth factors can hastenrecovery of some blood cell lineages, no therapy exists to treatsuppression of platelets, macrophages, T-cells or B-cells.

Roberts et al in 2012 reported that Pfizer compound PD-0332991 induced atransient cell cycle arrest in CDK4/6 dependent subsets of healthy cellssuch as HSPCs (see Roberts et al. Multiple Roles of Cyclin-DependentKinase 4/6 Inhibitors in Cancer Therapy. JNCI 2012; 104(6):476-487).This compound is currently being tested by Pfizer in clinical trials asan anti-neoplastic agent against estrogen-positive, HER2-negative breastcancer.

Hematopoietic stem cells give rise to progenitor cells which in turngive rise to all the differentiated components of blood as shown in FIG.1 (e.g., lymphocytes, erythrocytes, platelets, granulocytes, monocytes).HSPCs require the activity of CDK4/6 for proliferation (see Roberts etal. Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in CancerTherapy. JNCI 2012; 104(6):476-487). In healthy kidneys, the renalepithelium infrequently enters the cell cycle (about 1% of epithelialcells). After a renal insult, however, a robust increase in epithelialproliferation occurs (see Humphreys, B. D. et al. Intrinsic epithelialcells repair the kidney after injury. Cell Stem Cell 2, 284-91 (2008)).Importantly, following renal injury, surviving renal epithelial cellsreplicate to repair damage to the kidney tubular epithelium (seeHumphreys, B. D. et al. Repair of injured proximal tubule does notinvolve specialized progenitors. Proc Natl Acad Sci USA 108, 9226-31(2011)). See also WO 2010132725 filed by Sharpless et al.

A number of CDK 4/6 inhibitors have been identified, including specificpyrido[2,3-d]pyrimidines, 2-anilinopyrimidines, diaryl ureas,benzoyl-2,4-diaminothiazoles, indolo[6,7-a]pyrrolo[3,4-c]carbazoles, andoxindoles (see P. S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer DrugTargets 8 (2008) 53-75). WO 03/062236 identifies a series of2-(pyridin-2-ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment of Rbpositive cancers that show selectivity for CDK4/6, including6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one(PD0332991). The clinical trial studies have reported rates of Grade 3/4neutropenia and leukopenia with the use of PD0332991, resulting in 71%of patients requiring a dose interruption and 35% requiring a dosereduction; and adverse events leading to 10% of the discontinuations(see Finn, Abstract 51-6, SABCS 2012).

VanderWel et al. describe an iodine-containingpyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent and selective CDK4inhibitor (see VanderWel et al., J. Med. Chem. 48 (2005) 2371-2387).

WO 99/15500 filed by Glaxo Group Ltd discloses protein kinase andserine/threonine kinase inhibitors.

WO 2010/020675 filed by Novartis AG describes pyrrolopyrimidinecompounds as CDK inhibitors. WO 2011/101409 also filed by Novartisdescribes pyrrolopyrimidines with CDK 4/6 inhibitory activity.

WO 2005/052147 filed by Novartis and WO 2006/074985 filed by JanssenPharma disclose addition CDK4 inhibitors.

US 2007/0179118 filed by Barvian et al. teaches the use of CDK4inhibitors to treat inflammation.

WO 2012/061156 filed by Tavares and assigned to G1 Therapeuticsdescribes CDK inhibitors. WO 2013/148748 filed by Tavares and assignedto G1 Therapeutics describes Lactam Kinase inhibitors.

U.S. Patent Publication 2011/0224227 to Sharpless et al. describes theuse of certain CDK4/6 inhibitors, such as PD0332991 and 2BrIC (see Zhu,et al., J. Med. Chem., 46 (11) 2027-2030 (2003); PCT/US2009/059281) toreduce or prevent the effects of cytotoxic compounds on HSPCs in asubject undergoing chemotherapeutic treatments. See also U.S. PatentPublication 2012/0100100.

Stone, et al., Cancer Research 56, 3199-3202 (Jul. 1, 1996) describesreversible, p16-mediated cell cycle arrest as protection fromchemotherapy.

Accordingly, it is an object of the present invention to provide newcompounds, compositions and methods to treat patients duringchemotherapy.

SUMMARY OF THE INVENTION

In one embodiment, improved compounds, methods, and compositions areprovided to minimize the effect of chemotherapeutic agent toxicity onCDK4/6 replication dependent healthy cells, such as hematopoietic stemcells and hematopoietic progenitor cells (together referred to asHSPCs), and/or renal epithelial cells, in subjects, typically humans,that will be, are being, or have been exposed to the chemotherapeuticagent (typically a DNA-damaging agent).

Specifically, the invention includes administering an effective amountof a selected compound of Formula I, II, III, IV, or V, as describedherein, a pharmaceutically acceptable composition, salt, isotopicanalog, or prodrug thereof, which provides an optimal transientG1-arrest of healthy cells, for example HSPCs and/or renal epithelialcells, in a subject during or following the subject's exposure to achemotherapeutic agent, such as a DNA-damaging chemotherapeutic agent:

In one non-limiting example, a compound can be selected from thecompounds of Table 1 below, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof. In onenon-limiting example, a compound can be selected from compounds T, Q,GG, U, or AAAA, described below, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

The described compounds provide improved protection of CDK-replicationdependent healthy cells during chemotherapeutic agent treatment due inpart because they (i) exhibit a short, transient G1-arresting effect and(ii) display a rapid, synchronous reentry into the cell cycle by thecells following the cessation of the chemotherapeutic damaging effect.The use of these CDK4/6 specific, short, transient G1-arrestingcompounds as chemoprotectants allows for, for example, an acceleratedcell lineage recovery, reduced cytotoxicity risk due to replicationdelay, and/or a minimization of chemotherapeutic agent induced celldeath.

Despite reports using known CDK4/6 inhibitors such as 2BrIC andPD0332991 to demonstrate chemoprotection, it has been discovered thatthese inhibitors may not be the most ideal compounds for use inpharmacological quiescence (PQ) strategies. For example, the use of2BrIC in vivo is limited by its restricted bioavailability, and despitethe relative selectivity for CDK4/6 exhibited by PD0332991, the compoundhas a relatively long-acting intra-cellular effect (see Roberts et al.Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in CancerTherapy. JCNI 2012; 104(6):476-487 (FIG. 2A)), extending the transiencyof G1 arrest beyond what may be necessary for sufficient protection fromchemotherapeutic treatments. Such a long acting effect delays, forexample, the proliferation of HSPC cell lineages necessary toreconstitute the hematological cell lines that are adversely affected bychemotherapeutic agents or are cycled out during their naturallife-cycle. The long-acting G1 arrest provided by PD0332991 may limitits use as a potential chemoprotectant in subjects whosechemotherapeutic treatment regimen requires a rapid reentry into thecell cycle by HSPCs in order to reconstitute the erythroid, platelet,and myeloid cells (monocyte and granulocyte) adversely effected bychemotherapeutic agents or acute HSPC G1-arrest in order to limitmyelosuppressive or hematologic toxicity effects. Furthermore, PD0332991may be limited in its use as a chemoprotectant in subjects exposed tochemotherapeutic agents at regular and repeated intervals, for example,on regimens that are repeated every several days, as it may limit theability of these subjects' HSPCs to reenter the cell-cycle quicklybefore it would be necessary to arrest them again prior to the subject'snext chemotherapeutic cycle. With respect to other affected tissues, forexample renal cells, the timely resumption of proliferation is criticalto tissue repair, for example renal tubular epithelium repair, due tonephrotoxic agents, and therefore, an overly long period of PQ isundesirable.

Thus in an alternative embodiment, the invention includes administeringa compound described herein in an effective amount to a host in needthereof, such compound displaying one or any combination of thefollowing factors which provide an improved therapeutic effect (eitheralone or in any combination thereof, each of which is consideredspecifically and independently described): i) wherein a substantialportion of the CDK4/6-replication dependent healthy cells (e.g. at least80% or greater) return to or approach pre-treatment baseline cell cycleactivity (i.e., reenter the cell-cycle) in less than 24 hours, 30 hoursor 36 hours from the last administration of the active compound inhumans or for example, using a protocol described in the Examples below;ii) wherein a substantial portion of the healthy cells reenter thecell-cycle synchronously in less than 24 hours, 30 hours or 36 hoursfrom the last administration of the active compound; (iii) wherein thedissipation of the active compound's CDK4/6 inhibitory effect occurs inless than 24 hours, 30 hours, or 36 hours from the administration of theinhibitor; (iv) wherein the active compound has an IC50 for CDK4 and/orCDK6 inhibition that is more than 1500 times less than its IC50concentration for CDK2 inhibition; (v) wherein a substantial portion ofthe healthy cells return to or approach pre-treatment baseline cellcycle activity (i.e., reenter the cell-cycle) in less than 24 hours, 30hours, or 36 hours from the dissipation of the active compound's CDK4/6inhibitory effect; (vi) wherein the pre-treatment baseline cell cycleactivity (i.e. reenter the cell-cycle) within less than about 24 hours,about 30 hours, or about 36 hours from the point in which the CDK4/6inhibitor's concentration level in the subject's blood drops below atherapeutic effective concentration; or (vii) wherein a substantialportion of the healthy cells reenter the cell-cycle synchronously inless than 24 hours, 30 hours, or 36 hours from the last administrationof the chemotherapeutic agent.

The compounds described herein can be administered to the subject priorto treatment with a chemotherapeutic agent, during treatment with achemotherapeutic agent, after exposure to a chemotherapeutic agent, or acombination thereof. The compound described herein is typicallyadministered in a manner that allows the drug facile access to the bloodstream, for example via intravenous injection or sublingual,intraaortal, or other efficient blood-stream accessing route; however,oral, topical, transdermal, intranasal, intramuscular, or by inhalationsuch as by a solution, suspension, or emulsion, or other desiredadministrative routes can be used. In one embodiment, the compound isadministered to the subject less than about 24 hours, 20 hours, 16hours, 12 hours, 8 hours, or 4 hours, 2.5 hours, 2 hours, 1 hour, 12hour or less prior to treatment with the chemotherapeutic agent.Typically, the active compound described herein is administered to thesubject prior to treatment with the chemotherapeutic agent such that thecompound reaches peak serum levels before or during treatment with thechemotherapeutic agent. In one embodiment, the active compound isadministered concomitantly, or closely thereto, with thechemotherapeutic agent exposure. If desired, the active compound can beadministered multiple times during the chemotherapeutic agent treatmentto maximize inhibition, especially when the chemotherapeutic drug isadministered over a long period or has a long half-life. The activecompound described herein can be administered following exposure to thechemotherapeutic agent if desired to mitigate healthy cell damageassociated with chemotherapeutic agent exposure. In certain embodiments,the active compound is administered up to about 12 hour, up to about 1hour, up to about 2 hours, up to about 4 hours, up to about 8 hours, upto about 10 hours, up to about 12 hours, up to about 14 hours, up toabout 16 hours, or up to about 20 hours or greater following thechemotherapeutic agent exposure. In a particular embodiment, the activecompound is administered up to between about 12 hours and 20 hoursfollowing exposure to the chemotherapeutic agent.

The CDK4/6 inhibitors described herein show a marked selectivity for theinhibition of CDK4 and/or CDK6 in comparison to other CKD, for exampleCDK2. For example, CDK4/6 inhibitors described in the present inventionprovide for a dose-dependent G1-arresting effect on a subject'sCDK4/6-replication dependent healthy cells, for example HSPCs or renalepithelial cells, and the methods provided for herein are sufficient toafford chemoprotection to targeted CDk4/6-replication dependent healthycells during chemotherapeutic agent exposure, for example, during thetime period that a DNA-damaging chemotherapeutic agent is capable ofDNA-damaging effects on CDK4/6-replication dependent healthy cells inthe subject, while allowing for the synchronous and rapid reentry intothe cell-cycle by these cells shortly after the chemotherapeutic agentdissipates due to the time-limited CDK4/6 inhibitory effect provided bythe compounds described herein compared to, for example, PD0332991.Likewise, CDK4/6 inhibitors useful in the present invention provide fora dose-dependent mitigating effect on CDK4/6-replication dependenthealthy cells that have been exposed to toxic levels of chemotherapeuticagents, for example an accidental overdose, allowing for repair of DNAdamage associated with chemotherapeutic agent exposure and synchronous,rapid reentry into the cell-cycle following dissipation of the CDK4/6inhibitory effect compared to, for example, PD0332991. In oneembodiment, the use of a CDK4/6 inhibitor described herein results inthe G1-arresting effect on the subject's CDK4/6-replication dependenthealthy cells dissipating following administration of the CDK4/6inhibitor so that the subject's healthy cells return to or approachtheir pre-administration baseline cell-cycle activity within less thanabout 24 hours, 30 hours, 36 hours, or 40 hours, of administration. Inone embodiment, the G1-arresting effect dissipates such that thesubject's CDK4/6-replication dependent healthy cells return to theirpre-administration baseline cell-cycle activity within less than about24 hours, 30 hours, 36 hours, or 40 hours.

In one embodiment, the use of a CDK4/6 inhibitor described hereinresults in the G1-arresting effect dissipating such that the subject'sCDk4/6-dependent healthy cells return to or approach theirpre-administration baseline cell-cycle activity within less than about24 hours, 30 hours, 36 hours, or 40 hours of the chemotherapeutic agenteffect. In one embodiment, the G1-arresting effect dissipates such thatthe subject's CDK4/6-replication dependent cells return to theirpre-administration baseline cell-cycle activity within less than about24 hours, 30 hours, 36 hours, or 40 hours, or within about 48 hours ofthe cessation of the chemotherapeutic agent administration. In oneembodiment, the CDK4/6-replication dependent healthy cells are HSPCs. Inone embodiment, the CDK4/6-dependent healthy cells are renal epithelialcells.

In one embodiment, the use of a CDK4/6 inhibitor described hereinresults in the G1-arresting effect dissipating so that the subject'sCDK4/6-replication dependent healthy cells return to or approach theirpre-administration baseline cell-cycle activity within less than about24 hours, 30 hours, 36 hours, 40 hours, or within less than about 48hours from the point in which the CDK4/6 inhibitor's concentration levelin the subject's blood drops below a therapeutic effectiveconcentration.

In one embodiment, the CDK4/6 inhibitors described herein are used toprotect renal epithelium cells during exposure to a chemotherapeuticagent, for example, a DNA damaging chemotherapeutic agent, wherein therenal epithelial cells are transiently prevented from entering S-phasein response to chemotherapeutic agent induced renal tubular epitheliumdamage for no more than about 24 hours, about 30 hours, about 36 hours,about 40 hours, or about 48 hours from the point in which the CDK4/6inhibitor's concentration level in the subject's blood drops below atherapeutic effective concentration, from the cessation of thechemotherapeutic agent effect, or from administration of the CDK4/6administration.

CDK4/6 inhibitors useful in the described methods may be synchronous intheir off-effect, that is, upon dissipation of the G1 arresting effect,CDK4/6-replication dependent healthy cells exposed to a CDK4/6 inhibitordescribed herein reenter the cell-cycle in a similarly timed fashion.CDK4/6-replication dependent healthy cells that reenter the cell-cycledo so such that the normal proportion of cells in G1 and S arereestablished quickly and efficiently, within less than about 24 hours,30 hours, 36 hours, 40 hours, or within about 48 hours of the from thepoint in which the CDK4/6 inhibitor's concentration level in thesubject's blood drops below a therapeutic effective concentration.

This advantageously allows for a larger number of healthy cells to beginreplicating upon dissipation of the G1 arrest compared with asynchronousCDK4/6 inhibitors such as PD0332991.

In addition, synchronous cell-cycle reentry following G1 arrest using aCDK4/6 inhibitor described herein provides for the ability to time theadministration of hematopoietic growth factors to assist in thereconstitution of hematopoietic cell lines to maximize the growth factoreffect. As such, in one embodiment, the use of the compounds or methodsdescribed herein is combined with the use of hematopoietic growthfactors including, but not limited to, granulocyte colony stimulatingfactor (G-CSF), granulocyte-macrophage colony stimulating factor(GM-CSF), thrombopoietin, interleukin (IL)-12, steel factor, anderythropoietin (EPO), or their derivatives. In one embodiment, theCDK4/6 inhibitor is administered prior to administration of thehematopoietic growth factor. In one embodiment, the hematopoietic growthfactor administration is timed so that the CDK4/6 inhibitor's effect onHSPCs has dissipated.

In one aspect, the use of a CDK4/6-inhibitor described herein allows fora chemo-protective regimen for use during standard chemotherapeuticdosing schedules or regimens common in many anti-cancer treatments. Forexample, the CDK4/6-inhibitor can be administered so thatCDK4/6-replication dependent healthy cells are G1 arrested duringchemotherapeutic agent exposure wherein, due to the rapid dissipation ofthe G1-arresting effect of the compounds, a significant number ofhealthy cells reenter the cell-cycle and are capable of replicatingshortly after chemotherapeutic agent exposure, for example, within lessthan about 24, 30, 40, or 48 hours, and continue to replicate untiladministration of the CDK4/6-inhibitor in anticipation of the nextchemotherapeutic treatment. In one embodiment, the CDK4/6-inhibitor isadministered to allow for the cycling of the CDK4/6-replicationdependent healthy cells between G1-arrest and reentry into thecell-cycle to accommodate a repeated-dosing chemotherapeutic treatmentregimen, for example including but not limited to a treatment regimenwherein the chemotherapeutic agent is administered: on day 1-3 every 21days; on days 1-3 every 28 days; on day 1 every 3 weeks; on day 1, day8, and day 15 every 28 days, on day 1 and day 8 every 28 days; on days 1and 8 every 21 days; on days 1-5 every 21 days; 1 day a week for 6-8weeks; on days 1, 22, and 43; days 1 and 2 weekly; days 1-4 and 22-25;1-4; 22-25, and 43-46; and similar type-regimens, wherein theCDK4/6-replication dependent cells are G1 arrested duringchemotherapeutic agent exposure and a significant portion of the cellsreenter the cell-cycle between chemotherapeutic agent exposure. In oneembodiment, the CDK4/6-inhibitor can be administered so that thesubject's CDK4/6-replication dependent cells are G1-arrested duringdaily chemotherapeutic agent exposure, for example a contiguousmulti-day chemotherapeutic regimen, but a significant portion ofCDK4/6-replication dependent cells reenter the cell-cycle and replicatebetween daily treatment. In one embodiment, the CDK4/6-inhibitors can beadministered so that the subject's CDK4/6-replication dependent cellsare G1-arrested during chemotherapeutic agent exposure, for example acontiguous multi-day regimen, but a significant portion of healthy cellsreenter the cell-cycle and replicate during the off periods before thenext chemotherapeutic agent exposure. In one embodiment, the CDK4/6inhibitor is administered so that a subject's CDK4/6-replicationdependent cells' G1-arrest is provided during a daily chemotherapeuticagent treatment regimen, for example, a contiguous multi-day treatmentregimen, and the arrested cells are capable of reentering the cell-cycleshortly after the multi-day regimen ends. In one embodiment, the canceris small cell lung cancer and the CDK4/6 inhibitor is administered ondays 1, 2, and 3 during a 21-day treatment cycle wherein theadministered DNA damaging agent is selected from the group consisting ofcarboplatin, cisplatin, and etoposide, or a combination thereof.

The subject treated according to the present invention may be undergoingtherapeutic chemotherapy for the treatment of a proliferative disorderor disease such as cancer. The cancer can be characterized by one or acombination of increased activity of cyclin-dependent kinase 1 (CDK1),increased activity of cyclin-dependent kinase 2 (CDK2), loss,deficiency, or absence of retinoblastoma tumor suppressor protein(Rb)(Rb-null), high levels of MYC expression, increased cyclin E1, E2,and increased cyclin A. The cancer may be characterized by reducedexpression of the retinoblastoma tumor suppressor protein or aretinoblastoma family member protein or proteins (such as, but notlimited to p107 and p130). In one embodiment, the subject is undergoingchemotherapeutic treatment for the treatment of an Rb-null orRb-deficient cancer, including but not limited to small cell lungcancer, triple-negative breast cancer, HPV-positive head and neckcancer, retinoblastoma, Rb-negative bladder cancer, Rb negative prostatecancer, osteosarcoma, or cervical cancer. In one embodiment, the canceris a CDK4/6-independent cancer. Administration of the inhibitor compoundmay allow for a higher dose of a chemotherapeutic agent to be used totreat the disease than the standard dose that would be safely used inthe absence of administration of the CDK4/6 inhibitor compound.

The host or subject, including a human, may be undergoingchemotherapeutic treatment of a non-malignant proliferative disorder, orother abnormal cellular proliferation, such as a tumor, multiplesclerosis, lupus, or arthritis.

The protected HSPCs include hematopoietic stem cells, such as long termhematopoietic stem cells (LT-HSCs) and short term hematopoietic stemcells (ST-HSCs), and hematopoietic progenitor cells, includingmultipotent progenitors (MPPs), common myeloid progenitors (CMPs),common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors(GMPs) and megakaryocyte-erythroid progenitors (MEPs). Administration ofthe inhibitor compound provides temporary, transient pharmacologicquiescence of hematopoietic stem and/or hematopoietic progenitor cellsin the subject.

Administration of a CDK4/6 inhibitor as described herein can result inreduced anemia, reduced lymphopenia, reduced thrombocytopenia, orreduced neutropenia compared to that typically expected after, commonafter, or associated with treatment with chemotherapeutic agents in theabsence of administration of the CDK4/6 inhibitor. The use of the CDK4/6inhibitor as described herein results in a faster recovery from bonemarrow suppression associated with long-term use of CDK4/6 inhibitors,such as myelosuppression, anemia, lymphopenia, thrombocytopenia, orneutropenia, following the cessation of use of the CDK4/6 inhibitor. Insome embodiments, the use of a CDK4/6 inhibitor as described hereinresults in reduced or limited bone marrow suppression associated withlong-term use of CDK4/6 inhibitors, such as myelosuppression, anemia,lymphopenia, thrombocytopenia, or neutropenia.

In an alternative aspect, a CDK4/6 inhibitor described herein can beused for its anti-cancer, anti-tumor, or anti-proliferative effect incombination with a chemotherapeutic agent to treat an Rb-negative canceror other Rb-negative abnormal proliferation. In one embodiment, theCDK4/6 inhibitor described herein provides an additive effect to orsynergistic effect with the anti-cancer or anti-proliferative activityof the chemotherapeutic. Chemotherapeutics that can be combined with theCDK4/6 inhibitors described herein are any chemotherapeutics effectiveor useful to treat RB-null cancers or abnormal cellular proliferation.In one particular embodiment, the use of a compound described herein iscombined in a therapeutic regime with at least one otherchemotherapeutic agent, and can be one that does not rely onproliferation or advancement through the cell-cycle foranti-proliferative activity. Such agent may include, but is not limitedto, tamoxifen, midazolam, letrozole, bortezomib, anastrozole, goserelin,an mTOR inhibitor, a PI3 kinase inhibitors, dual mTOR-PI3K inhibitors,MEK inhibitors, RAS inhibitors, ALK inhibitors, HSP inhibitors (forexample, HSP70 and HSP 90 inhibitors, or a combination thereof), BCL-2inhibitors, apopototic inducing compounds, AKT inhibitors, PD-1inhibitors, or FLT-3 inhibitors, or combinations thereof. Examples ofmTOR inhibitors include but are not limited to rapamycin and itsanalogs, everolimus (Afinitor), temsirolimus, ridaforolimus, sirolimus,and deforolimus. Examples of P13 kinase inhibitors include but are notlimited to Wortmannin, demethoxyviridin, perifosine, idelalisib, PX-866,IPI-145 (Infinity), BAY 80-6946, BEZ235, RP6503, TGR 1202 (RP5264),MLN1117 (INK1117), Pictilisib, Buparlisib, SAR245408 (XL147), SAR245409(XL765), Palomid 529, ZSTK474, PWT33597, RP6530, CUDC-907, and AEZS-136.Examples of MEK inhibitors include but are not limited to Tametinib,Selumetinib, MEK162, GDC-0973 (XL518), and PD0325901. Examples of RASinhibitors include but are not limited to Reolysin and siG12D LODER.Examples of ALK inhibitors include but are not limited to Crizotinib,AP26113, and LDK378. HSP inhibitors include but are not limited toGeldanamycin or 17-N-Allylamino-17-demethoxygeldanamycin (17AAG), andRadicicol. The CDK4/6 inhibitor combined with the chemotherapeutic isselected from the group consisting of a compound or compositioncomprising Formula I, Formula II, Formula III, Formula IV, or Formula Vdescribed above, or a pharmaceutically acceptable composition, salt,isotopic analog or prodrug thereof. In one embodiment, the compound isselected from the compounds provided for in Table 1, or apharmaceutically acceptable composition, salt, isotopic analog orprodrug thereof. In one embodiment, the compound is selected fromcompounds T, Q, GG, U, or AAAA, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

In certain embodiments, a compound described herein is administered tothe subject prior to treatment with another chemotherapeutic agent,during treatment with another chemotherapeutic agent, afteradministration of another chemotherapeutic agent, or a combinationthereof. In one embodiment, the CDK4/6 inhibitor is selected from acompound described in Table 1. In one embodiment, the compound isselected from compounds T, Q, GG, U, or AAAA.

In some embodiments, the subject or host is a mammal, including a human.

In summary, the present invention includes the following features:

-   -   A. Compounds of Formula I, II, III, IV, and V as described        herein, and pharmaceutically acceptable compositions, salts,        isotopic analogs, or prodrugs thereof, for use in the        chemoprotection of CDK4/6-replication dependent healthy cells,        for example HSPCs and/or renal epithelial cells, during a        chemotherapeutic agent exposure. In one embodiment, the compound        is selected from the compounds described in Table 1 or a        pharmaceutically acceptable composition, salt, isotopic analog        or prodrug thereof. In one embodiment, the compound is selected        from compounds T, Q, GG, U, or AAAA, or a pharmaceutically        acceptable composition, salt, isotopic analog or prodrug        thereof;    -   B. Compounds of Formula I, II, III, IV, and V as described        herein, and pharmaceutically acceptable compositions, salts,        isotopic analogs, and prodrugs thereof, for use in the        chemoprotection of CDK4/6-replication dependent healthy cells,        for example HSPCs and/or renal epithelial cells, during a        chemotherapeutic regimen for the treatment of a proliferative        disorder. In one embodiment, the compound is selected from the        compounds described in Table 1 or a pharmaceutically acceptable        composition, salt, isotopic analog or prodrug thereof. In one        embodiment, the compound is selected from compounds T, Q, GG, U,        or AAAA, or a pharmaceutically acceptable composition, salt,        isotopic analog or prodrug thereof;    -   C. Compounds of Formula I, II, III, IV, and V as described        herein, and pharmaceutically acceptable compositions, salts,        isotopic analogs, and prodrugs thereof, for use in the        chemoprotection of CDK4/6-replication dependent healthy cells,        for example HSPCs and/or renal epithelial cells, during a        chemotherapeutic regimen for the treatment of a cancer. In one        embodiment, the compound is selected from the compounds        described in Table 1, or a pharmaceutically acceptable        composition, salt, isotopic analog or prodrug thereof. In one        embodiment, the compound is selected from compounds T, Q, GG, U,        or AAAA, or a pharmaceutically acceptable composition, salt,        isotopic analog or prodrug thereof;    -   D. Compounds of Formula I, II, III, IV, and V as described        herein, and pharmaceutically acceptable compositions, salts,        isotopic analogs, and prodrugs thereof, for use in combination        with hematopoietic growth factors in a subject that will be, is        being, or has been exposed to chemotherapeutic agents. In one        embodiment, the compound is selected from the compounds        described in Table 1 or a pharmaceutically acceptable        composition, salt, isotopic analog or prodrug thereof. In one        embodiment, the compound is selected from compounds T, Q, GG, U,        or AAAA, or a pharmaceutically acceptable composition, salt,        isotopic analog or prodrug thereof;    -   E. Use of compounds of Formula I, II, III, IV, and V as        described herein, and pharmaceutically acceptable compositions,        salts, isotopic analogs, and prodrugs thereof, in the        manufacture of a medicament for use in the chemoprotection of        CDK4/6-replication dependent healthy cells, for example HSPCs        and/or renal epithelial cells. In one embodiment, the compound        is selected from the compounds described in Table 1 or a        pharmaceutically acceptable composition, salt, isotopic analog        or prodrug thereof. In one embodiment, the compound is selected        from compounds T, Q, GG, U, or AAAA, or a pharmaceutically        acceptable composition, salt, isotopic analog or prodrug        thereof,    -   F. Use of compounds of Formula I, II, III, IV, and V as        described herein, and pharmaceutically acceptable compositions,        salts, isotopic analogs, and prodrugs thereof, in the        manufacture of a medicament for use in the mitigation of DNA        damage of CDK4/6-replication dependent healthy cells, for        example HSPCs and/or renal epithelial cells, that have been        exposed to chemotherapeutic agent exposure. In one embodiment,        the compound is selected from the compounds described in Table 1        or a pharmaceutically acceptable composition, salt, isotopic        analog or prodrug thereof. In one embodiment, the compound is        selected from compounds T, Q, GG, U, or AAAA, or a        pharmaceutically acceptable composition, salt, isotopic analog        or prodrug thereof;    -   G. A pharmaceutical formulation comprising an effective        subject-treating amount of compounds of Formula I, II, III, IV,        and V as described herein, or pharmaceutically acceptable        compositions, salts, and prodrugs thereof for use in        chemoprotection of healthy cells. In one embodiment, the        compound is selected from the compounds described in Table 1 or        a pharmaceutically acceptable composition, salt, isotopic analog        or prodrug thereof. In one embodiment, the compound is selected        from compounds T, Q, GG, U, or AAAA, or a pharmaceutically        acceptable composition, salt, isotopic analog or prodrug        thereof;    -   H. A processes for the preparation of therapeutic products that        contain an effective amount of compounds of Formula I, II, III,        IV, and V as described herein. In one embodiment, the compound        is selected from the compounds described in Table 1 or a        pharmaceutically acceptable composition, salt, isotopic analog        or prodrug thereof. In one embodiment, the compound is selected        from compounds T, Q, GG, U, or AAAA, or a pharmaceutically        acceptable composition, salt, isotopic analog or prodrug        thereof,    -   I. A method for manufacturing a medicament of Formula I, II,        III, IV, and V intended for therapeutic use in the        chemoprotection of CDK4/6-replication dependent healthy cells,        for example HSPCs and/or renal epithelial cells. In one        embodiment, the medicament is selected from the compounds        described in Table 1 or a pharmaceutically acceptable        composition, salt, isotopic analog or prodrug thereof. In one        embodiment, the medicament is selected from compounds T, Q, GG,        U, or AAAA, or a pharmaceutically acceptable composition, salt,        isotopic analog or prodrug thereof;    -   J. A method for manufacturing a medicament of Formula I, II,        III, IV, and V intended for therapeutic use in the mitigation of        DNA damage of CDK4/6-replication dependent healthy cells, for        example HSPCs and/or renal epithelial cells, that have been        exposed to chemotherapeutic agents. In one embodiment, the        medicament is selected from the compounds described in Table 1        or a pharmaceutically acceptable composition, salt, isotopic        analog or prodrug thereof. In one embodiment, the medicament is        selected from Compounds T, Q, GG, U, or AAAA, or a        pharmaceutically acceptable composition, salt, isotopic analog        or prodrug thereof;    -   K. A method of inhibiting the growth of an Rb-negative cancer or        proliferative condition by administering a compound of Formula        I, II, III, IV, or V, or pharmaceutically acceptable        composition, salt, isotopic analog or prodrug thereof; in        combination with a chemotherapeutic to provide an additive to or        synergistic effect with a chemotherapeutic. In one embodiment,        the compound is selected from the compounds described in Table 1        or a pharmaceutically acceptable composition, salt, isotopic        analog or prodrug thereof. In one embodiment, the compound is        selected from Compounds T, Q, GG, U, or AAAA, or a        pharmaceutically acceptable composition, salt, isotopic analog        or prodrug thereof. In one embodiment, the CDK4/6 inhibitors are        combined with a chemotherapeutic selected from the group        consisting of MEK inhibitors, PI3 kinase delta inhibitors, BCL-2        inhibitors, AKT inhibitors, apoptotic inducing compounds, AKT        inhibitors, PD-1 inhibitors, FLT-3 inhibitors, HSP90 inhibitors,        or mTOR inhibitors, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of hematopoiesis showing the hierarchicalproliferation of healthy hematopoietic stem cells (HSC) and healthyhematopoietic progenitor cells with increasing differentiation uponproliferation.

FIG. 2A is a graph of the percentage of cells in G2-M phase (opencircles), S phase (triangles), G0-G1 phase (squares), <2N (diamonds) vs.variable concentration (nM) of Compound T in tHS68 cells. TheCdk4/6-dependent cell line (tHS68) was treated with the indicatedconcentrations of Compound T for 24 hours. Following treatment ofCompound T, cells were harvested and analyzed for cell cycledistribution. As described in Example 152, tHS68 cells show a clean G1arrest accompanied by a corresponding decrease in the number of cells inS-phase. FIG. 2B is a graph of the number of tHS68 cells(CDK4/6-dependent cell line) vs. the DNA content of the cells (asmeasured by propidium iodide). Cells were treated with DMSO for 24hours, harvested, and analyzed for cell cycle distribution. FIG. 2C is agraph of the number of WM2664 cells (CDK4/6-dependent cell line) vs. theDNA content of the cells (as measured by propidium iodide). Cells weretreated with DMSO for 24 hours, harvested, and analyzed for cell cycledistribution. FIG. 2D is a graph of the number of A2058 cells(CDK4/6-independent cell line) vs. the DNA content of the cells (asmeasured by propidium iodide). Cells were treated with DMSO for 24hours, harvested, and analyzed for cell cycle distribution. FIG. 2E is agraph of the number of tHS68 cells (CDK4/6-dependent cell line) vs. theDNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 152, treatment of tHS68 cells with Compound Tcauses a loss of the S-phase peak (indicated by arrow). FIG. 2F is agraph of the number of WM2664 cells (CDK4/6-dependent cell line) vs. theDNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 152, treatment of WM2664 cells with Compound Tcauses a loss of the S-phase peak (indicated by arrow). FIG. 2G is agraph of the number of A2058 cells (CDK4/6-independent cell line) vs.the DNA content of the cells (as measured by propidium iodide) aftertreatment with Compound T. Cells were treated with Compound T (300 nM)for 24 hours, harvested, and analyzed for cell cycle distribution. Asdescribed in Example 152, treatment of A2058 cells with Compound T doesnot cause a loss of the S-phase peak (indicated by arrow).

FIG. 3 is a Western blot showing the phosphorylation levels of Rb atSer807/811 and Ser780 after treatment with Compound T. Cdk4/6-dependent(tHS68 or WM2664) and Cdk4/6-independent cell lines (A2058) were treatedwith Compound T (300 nM) for the indicated times (0, 4, 8, 16, and 24hours). MAPK levels are shown as a control for protein levels. Followingtreatment, cells were harvested and analyzed for Rb-phosphorylation bywestern blot analysis. As described in Example 153, Compound T treatmentresulted in reduced Rb-phosphorylation after treatment inCdk4/6-dependent cell lines (tHS68 and WM2664), but not in theCdk4/6-independent cell line (A2058).

FIG. 4A is a graph of the percentage of cells in S phase in anRb-positive cell line (WM2664) or in the Rb-negative small cell lungcancer cell lines (H345, H69, H209, SHP-77, NCI417, or H82) aftertreatment with DMSO (dark bars) or PD0332991 (light bars). Cells weretreated with PD0332991 (300 nM) or DMSO control for 24 hours. Cellproliferation was measured by EdU incorporation and flow cytometry. Datarepresents 100,000 cell events for each cell treatment. As described inExample 154, the RB-null SCLC cell line was resistant to Cdk4/6inhibition, as no changes in the percent of cells in S-phase were seenupon treatment with PD0332991.

FIG. 4B is a graph of the percentage of cells in S phase in anRb-positive cell line (tHS68) or in the Rb-negative small cell lungcancer cell lines (H345, H69, SHP-77, or H82) after treatment with DMSO(dark bars) or Compound GG (lighter bars). Cells were treated withCompound GG (300 nM or 1000 nM) or DMSO control for 24 hours. Cellproliferation was measured by EdU incorporation and flow cytometry. Datarepresents 100,000 cell events for each cell treatment. As described inExample 154, the RB-null SCLC cell line was resistant to Cdk4/6inhibition, as no changes in the percent of cells in S-phase were seenupon treatment with Compound GG.

FIG. 4C is a graph of the percentage of cells in S phase in anRb-positive cell line (tHS68) or in the Rb-negative small cell lungcancer cell lines (H345, H209, or SHP-77) after treatment with DMSO(dark bars) or Compound T (lighter bars). Cells were treated withCompound T (300 nM or 1000 nM) or DMSO control for 24 hours. Cellproliferation was measured by EdU incorporation and flow cytometry. Datarepresents 100,000 cell events for each cell treatment. As described inExample 154, the RB-null SCLC cell line was resistant to Cdk4/6inhibition, as no change in the percent of cells in S-phase were seenupon treatment with Compound T.

FIG. 5 is a graph of EdU incorporation vs. time after administration(hours) of PD0332991 to healthy mice HSPCs and healthy myeloidprogenitor cells. PD0332991 (150 mg/kg) was administered by oral gavageto assess the temporal effect of transient CDK4/6 inhibition on bonemarrow arrest as reported in Roberts et al. Multiple Roles ofCyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy. JCNI 2012;104(6):476-487 (FIG. 2A). As described in Example 156, a single oraldose of PD0332991 results in a sustained reduction in HSPC EdUincorporation (circles; LKS+) and myeloid progenitor cells EdUincorporation (squares; LKS−) for greater than 36 hours.

FIG. 6A is a graph of the ratio of EdU incorporation into HSPCs(compared to untreated control mice) following oral gavage of CompoundsT, Q, or GG at 150 mg/kg at either 12 or 24 hours post administration.FIG. 6B is a graph of the percentage of EdU positive HSPC cells for micetreated with Compound T at either 12 or 24 hours. Mice were dosed with50 mg/kg (triangles), 100 mg/kg (squares), or 150 (upside downtriangles) mg/kg by oral gavage. FIG. 6C is a graph of the percentage ofEdU positive HSPC cells for mice treated with compound T (150 mg/kg byoral gavage) at either 12, 24, 36 and 48 hours. As described in Example157, Compound T and GG demonstrated a reduction in EdU incorporation at12 hours, and started to return to normal levels of cell division by 24hours.

FIG. 7 is a graph of the percentage of EdU positive HSPC cells for micetreated with either PD0332991 (triangles) or Compound T (upside downtriangles) v. time after administration (hours) of the compound. Bothcompounds were administered at 150 mg/kg by oral gavage and thepercentage of EdU positive HSPC cells was measured at 12, 24, 36 or 48hours. As described in Example 158, a single oral dose of PD0332991results in a sustained reduction of HSPC proliferation for greater than36 hours. In contrast, a single oral dose of Compound T results in aninitial reduction of HSPC proliferation at 12 hours, but proliferationof HSPCs resumes by 24 hours after dosage of Compound T.

FIG. 8A is a graph of the percentage of cells in the G0-G1 phase of thecell cycle vs. time after washout of the compound (hours) in humanfibroblast (Rb-positive) cells. FIG. 8B is a graph of the percentage ofcells in the S phase of the cell cycle vs. time after washout of thecompound (hours) in human fibroblast (Rb-positive) cells. FIG. 8C is agraph of the percentage of cells in the G0-G1 phase of the cell cyclevs. time after washout of the compound (hours) in human renal proximaltubule epithelial (Rb-positive) cells. FIG. 8D is a graph of thepercentage of cells in the S phase of the cell cycle vs. time afterwashout of the compound (hours) in human renal proximal tubuleepithelial (Rb-positive) cells. These cellular wash out experimentsdemonstrated that the inhibitor compounds of the present invention havea short, transient G1-arresting effect in different cell types. Theeffect on the cell cycle following washing out of the compounds wasdetermined at 24, 36, 40, and 48 hours. As described in Example 159, theresults show that cells treated with PD0332991 (circles) tooksignificantly longer to reach baseline levels of cell division (seecells treated only with DMSO (diamonds)), than cells treated withCompound T (squares), Compound Q (triangles), Compound GG (X), orCompound U (X with cross).

FIG. 9A is a graph of plasma drug concentration (ng/ml) vs. time afteradministration (hours) of Compound T. FIG. 9B is a graph of plasma drugconcentration (ng/ml) vs. time after administration (hours) of CompoundQ. FIG. 9C is a graph of plasma drug concentration (ng/ml) vs. timeafter administration (hours) of compound GG. FIG. 9D is a graph ofplasma drug concentration (ng/ml) vs. time after administration (hours)of Compound U. Compounds were dosed to mice at 30 mg/kg by oral gavage(diamonds) or 10 mg/kg by intravenous injection (squares). Blood sampleswere taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours post dosing andthe plasma concentrations were determined by HPLC.

FIG. 10 provides the half-life (minutes) of Compound T and PD0332991 inhuman and animal (monkey, dog, rat, and mouse) liver microsomes. Asdescribed in Example 158, PD0332991 has a half-life greater than 60minutes in each of the species tested. Compound T was determined to havea shorter half-life than PD0332991 in each of the species tested.

FIG. 11A is a graph of cell survival of cells treated with 5 uMetoposide vs. treatment with the indicated amount of Compound T.Surviving cells were determined at 24 hours post treatment. As describedin Example 162, shows that Compound T protects tHS68 cells fromchemotherapeutic induced cell death. FIG. 11B is a graph of cellsurvival of cells treated with 100 μM carboplatin vs. treatment with theindicated amount of Compound T. Surviving cells were determined at 24hours post treatment. As described in Example 162, Compound T protectstHS68 cells from chemotherapeutic induced cell death.

FIG. 12A is a graph of the relative H2AX activity vs. variableconcentration of Compound T (nM) in HS68 cells treated with Compound T(100 nM, 300 nM, or 1000 nM) and chemotherapy (etoposide, doxorubicin,carboplatin, paclitaxel, or camptothecin). Cdk4/6-dependent HS68 cellswere treated with the indicated doses of Compound T and chemotherapy.H2AX foci formation was measured to evaluate chemotherapy-induced DNAdamage. As described in Example 162, cells treated with Compound T andvarious chemotherapeutic compounds were protected from DNA damageinduced by the chemotherapy.

FIG. 12B is a graph of the relative Caspase 3/7 activity vs. variableconcentration of Compound T (nM) in HS68 cells treated with Compound T(100 nM, 300 nM, or 1000 nM) and chemotherapy (etoposide, doxorubicin,carboplatin, paclitaxel, or camptothecin). Cdk4/6-dependent HS68 cellswere treated with the indicated doses of Compound T and chemotherapy.Caspase 3/7 activity was measured to evaluate chemotherapy-inducedapoptosis. As described in Example 162, cells treated with Compound Tand various chemotherapeutic compounds were protected from caspase 3/7activation induced by the chemotherapy.

FIG. 13 is a series of contour plots showing proliferation (as measuredby EdU incorporation after 12 hours) vs. cellular DNA content (asmeasured by DAPI staining). Representative contour plots showproliferation in WBM (whole bone marrow; top) and HSPCs (hematopoieticstem and progenitor cells; LSK; bottom), as measured by EdUincorporation after 12 hours of no treatment, EdU treatment only, or EdUplus Compound T treatment. As described in Example 163, Compound Treduces proliferation of whole bone marrow and hematopoietic stem and/orprogenitor cells.

FIG. 14A is a graph of the percentage of EdU-positive cells in wholebone marrow (WBM) and various hematopoietic stem and progenitor cells(Lin−, LSK, HSC, MPP, or CD28+LSK cell lineages) treated with Compound T(open bars) or untreated (solid bars). As described in Example 163,treatment with Compound T inhibits proliferation of WBM and all HSPClineages tested. *P<0.05, **P<0.01.

FIG. 14B is a graph of the percentage of EdU-positive cells in wholebone marrow (WBM) and various lineage restricted progenitors (MP, GMP,MEP, CMP, or CLP cell lineages) treated with Compound T (open bars) oruntreated (solid bars). As described in Example 163, treatment withCompound T inhibits proliferation of WBM and all lineage restrictedprogenitors tested. *P<0.05, **P<0.01.

FIG. 15A is a graph of the percentage of EdU-positive cells in T cellpopulations (Total, CD4+, CD8+, DP, DN, DN1, DN2, DN3, or DN4) treatedwith Compound T (open bars) or untreated (solid bars). As described inExample 164, treatment with Compound T inhibits proliferation of theCD4+, CD8+, DP, DN, DN1, DN2, DN3, or DN4 T cell populations. *P<0.05,**P<0.01.

FIG. 15B is a graph of the percentage of EdU-positive cells in B cellpopulations (B220+, B220+ sIgM+, Pre-pro-B sIgM−, Pro-B, Pre-B) treatedwith Compound T (open bars) or untreated (solid bars). As described inExample 164, treatment with Compound T inhibits proliferation of thevarious B cell populations (B220+, B220+ sIgM+, Pre-pro-B sIgM−, Pro-B,and Pre-B). *P<0.05, **P<0.01.

FIG. 15C is a graph of the percentage of EdU-positive cells in myeloidcell populations (Mac1+Gr1+, Ter119+, or CD41+) treated with Compound T(open bars) or untreated (solid bars). As described in Example 164,treatment with Compound T inhibits proliferation of the Mac1+Gr1+,Ter119+, or CD41+ myeloid cell populations. *P<0.05, **P<0.01.

FIG. 16 shows the pharmacodynamic assessment of Compound GG in the bonemarrow. To assess the effect of transient CDK4/6 inhibition by CompoundGG on carboplatin-induced cytotoxicity in the bone marrow, FVB/n mice(n=3 per group) were treated with vehicle control, 90 mg/kg carboplatinby intraperitoneal injection, or 150 mg/kg Compound GG by oral gavageplus 90 mg/kg carboplatin by intraperitoneal injection. 24 hours aftertreatment bone marrow was harvested and the percent of cycling bonemarrow progenitors was measured by EdU incorporation as explainedearlier.

FIG. 17A is a graph of whole blood cell counts vs. time (days) afteradministration of 5-fluoruracil (5FU) (triangles), 5FU plus Compound T(squares), or untreated control (circles). FVB wild-type mice weretreated with Compound T (150 mg/kg) or vehicle control by oral gavagethirty minutes prior to administration of 5-flurouracil (5FU) 150 mg/kgby intraperitoneal injection. Complete blood cell counts were measuredevery two days starting on day six. As described in Example 166, wholeblood cells recover more rapidly from chemotherapy (5FU) when pretreatedwith Compound T. FIG. 17B is a graph of neutrophil cell counts vs. time(days) after administration of 5-fluoruracil (5FU) (triangles), 5FU plusCompound T (squares), or untreated control (circles). Experiments wereconducted as described in FIG. 17A. As described in Example 166,neutrophils recover more rapidly from chemotherapy (5FU) when pretreatedwith Compound T. FIG. 17C is a graph of lymphocyte cell counts vs. time(days) after administration of 5-fluoruracil (5FU) (triangles), 5FU plusCompound T (squares), or untreated control (circles). Experiments wereconducted as described in FIG. 17A. As described in Example 166,lymphocytes recover more rapidly from chemotherapy (5FU) when pretreatedwith Compound T. FIG. 17D is a graph of platelet cell counts vs. time(days) after administration of 5-fluoruracil (5FU) (triangles), 5FU plusCompound T (squares), or untreated control (circles). Experiments wereconducted as described in FIG. 17A. As described in Example 166,platelets recover more rapidly from chemotherapy (5FU) when pretreatedwith Compound T. FIG. 17E is a graph of red blood cell counts vs. time(days) after administration of 5-fluoruracil (5FU) (triangles), 5FU plusCompound T (squares), or untreated control (circles). Experiments wereconducted as described in FIG. 17A. As described in Example 166, redblood cells recover more rapidly from chemotherapy (5FU) when pretreatedwith Compound T. FIG. 17F is a graph of hematocrit (%) vs. time (days)after administration of 5-fluoruracil (5FU) (triangles), 5FU plusCompound T (squares), or untreated control (circles). Experiments wereconducted as described in FIG. 17A. As described in Example 166,hematocrit percentage recovers more rapidly from chemotherapy (5FU) whenpretreated with Compound T.

FIG. 18A is a graph of whole blood cell counts 14 days afteradministration of 5-fluoruracil (5FU), 5FU plus Compound T, or untreatedcontrol. FVB wild-type mice were treated with Compound T (150 mg/kg) orvehicle control by oral gavage thirty minutes prior to administration of5-flurouracil (5FU) 150 mg/kg by intraperitoneal injection. Completeblood cell counts were measured on day 14. Boxes represent the 5%-95%distribution, whiskers represent minimum and maximum values, and themiddle bar represents the median. Student's t test was done to calculatetwo-sided P values. As described in Example 166, whole blood cellsrecover more rapidly from chemotherapy (5FU) when pretreated withCompound T. FIG. 18B is a graph of neutrophil cell counts 14 days afteradministration of 5-fluoruracil (5FU), 5FU plus Compound T, or untreatedcontrol. Experiments were conducted as described in FIG. 18A. Asdescribed in Example 166, neutrophil cells recover more rapidly fromchemotherapy (5FU) when pretreated with Compound T. FIG. 18C is a graphof lymphocyte cell counts 14 days after administration of 5-fluoruracil(5FU), 5FU plus Compound T, or untreated control. Experiments wereconducted as described in FIG. 18A. As described in Example 166,lymphocyte cells recover more rapidly from chemotherapy (5FU) whenpretreated with Compound T. FIG. 18D is a graph of red blood cell counts14 days after administration of 5-fluoruracil (5FU), 5FU plus CompoundT, or untreated control. Experiments were conducted as described in FIG.18A. As described in Example 166, red blood cells recover more rapidlyfrom chemotherapy (5FU) when pretreated with Compound T. FIG. 18E is agraph of platelet cell counts 14 days after administration of5-fluoruracil (5FU), 5FU plus Compound T, or untreated control.Experiments were conducted as described in FIG. 18A. As described inExample 166, platelet cells recover more rapidly from chemotherapy (5FU)when pretreated with Compound T.

FIG. 19A is a graph of whole blood cells (1000 cells/ul) in untreatedmice (circles), 5-fluoruracil (5FU) plus Compound T treated mice(squares), or 5-FU treated mice (triangles) at Cycle 3, Day 10 (Day 52).FVB wild-type mice were treated with Compound T (150 mg/kg) or vehiclecontrol by oral gavage thirty minutes prior to administration of5-flurouracil (5FU) 150 mg/kg by intraperitoneal injection. Micereceived 3 cycles of Compound T or vehicle control+5FU on Day 1 of a21-day cycle. Complete blood cell counts were measured on Day 10 afterthe second dose (52 days after the first dose (Cycle 3 Day 10)). Asdescribed in Example 167, whole blood cells show an improved recoveryfrom chemotherapy (5FU) when treated with several cycles of Compound T.FIG. 19B is a graph of neutrophils (1000 cells/ul) in untreated mice(circles), 5-fluoruracil (5FU) plus Compound T treated mice (squares),or 5-FU treated mice (triangles) at Cycle 3, Day 10 (Day 52).Experiments were conducted as described in FIG. 19A. As described inExample 167, neutrophils show an improved recovery from chemotherapy(5FU) when treated with several cycles of Compound T. FIG. 19C is agraph of lymphocytes (1000 cells/ul) in untreated mice (circles),5-fluoruracil (5FU) plus Compound T treated mice (squares), or 5-FUtreated mice (triangles) at Cycle 3, Day 10 (Day 52). Experiments wereconducted as described in FIG. 19A. As described in Example 167,lymphocytes show an improved recovery from chemotherapy (5FU) whentreated with several cycles of Compound T. FIG. 19D is a graph of redblood cells (1000 cells/ul) in untreated mice (circles), 5-fluoruracil(5FU) plus Compound T treated mice (squares), or 5-FU treated mice(triangles) at Cycle 3, Day 10 (Day 52). Experiments were conducted asdescribed in FIG. 19A. As described in Example 167, red blood cells showan improved recovery from chemotherapy (5FU) when treated with severalcycles of Compound T. FIG. 19E is a graph of platelets (1000 cells/ul)in untreated mice (circles), 5-fluoruracil (5FU) plus Compound T treatedmice (squares), or 5-FU treated mice (triangles) at Cycle 3, Day 10 (Day52). Experiments were conducted as described in FIG. 19A. As describedin Example 167, platelet levels are elevated in recovery fromchemotherapy (5FU) when treated with several cycles of Compound T.

FIG. 20 is a graph of the percentage of cells in G2-M phase (X), S phase(triangles), G0-G1 phase (squares), or <2N (diamonds) vs. variableconcentration (nM) of compound T in human renal proximal tubule cells.The cells were treated with the indicated concentrations of Compound Tfor 24 hours. Following treatment of Compound T, cells were harvestedand analyzed for cell cycle distribution. As described in Example 168,human renal proximal tubule cells show a clean G1 arrest accompanied bya corresponding decrease in the number of cells in S-phase.

FIG. 21 is a graph of the percentage of cells in G2-M phase (X), S phase(triangles), G0-G1 phase (squares), or <2N (diamonds) vs. variableconcentration (nM) of Compound T in human renal proximal tubule cellstreated with DMSO, etoposide, or cisplatin. The cells were treated withthe indicated concentrations of Compound T for 24 hours in combinationwith DMSO, etoposide, or cisplatin. Following treatment of Compound T,cells were harvested and analyzed for cell cycle distribution. Asdescribed in Example 169, treatment of human renal proximal tubule cellswith Compound T protects these cells from chemotherapy induced damage byetoposide and cisplatin.

FIG. 22 is a graph of the relative γ-H2AX activity vs. variableconcentration of Compound T (nM) in human renal proximal tubule cellstreated with Compound T and chemotherapy (cisplatin). Cells were treatedwith the indicated doses of Compound T (10 nM, 30 nM, 100 nM, 300 nM, or1000 nM) and chemotherapy (25 uM cisplatin). γ-H2AX foci formation wasmeasured to evaluate chemotherapy-induced DNA damage. As described inExample 170, cells treated with Compound T were protected from DNAdamage induced by the chemotherapy (cisplatin).

FIG. 23 is a graph of caspase 3/7 activation (as measured by relativelight units) in renal tubule epithelial cells treated with the indicatedconcentrations of Compound T and either DMSO or cisplatin (25 uM, 50 uM,or 100 uM). Normal renal proximal tubule epithelial cells were obtainedfrom American Type Culture Collection (ATCC, Manassas, VA). Cells weregrown in an incubator at 37° C. in a humidified atmosphere of 5% CO2 inRenal Epithelial Cell Basal Media (ATCC) supplemented with RenalEpithelial Cell Growth Kit (ATCC) in 37° C. humidified incubator. Cellswere treated with DMSO or 30 nM, 100 nM, 300 nM or 1 uM Compound T ineither the absence or presence of 25, 50 uM, or 100 uM cisplatin.Caspase 3/7 activation was measured 24 hours later using the Caspase-Glo3/7 Assay System (Promega, Madison, WI) by following the manufacturer'sinstructions. As described in Example 170, Compound T demonstrated adose-dependent reduction in caspase 3/7 activation in these cells.

FIGS. 24-26 illustrate several exemplary embodiments of R² of thecompounds of the invention.

FIGS. 27A-27C, 28A-D, 29A-29C, 30A-30B, and 31A-31F illustrate exemplaryembodiments of the core structure of the compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Improved compounds, methods, and compositions are provided to minimizethe effect of chemotherapeutic agent toxicity on CDK4/6 replicationdependent healthy cells, such as hematopoietic stem cells and/orhematopoietic progenitor cells (together referred to as HSPCs), and/orrenal epithelial cells, in subjects, typically humans, that will be, arebeing or have been exposed to the chemotherapeutic agent (typically aDNA-damaging agent).

Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Definition of standard chemistryterms may be found in reference works, including Carey and Sundberg(2007) Advanced Organic Chemistry 5^(th) Ed. Vols. A and B, SpringerScience+Business Media LLC, New York. The practice of the presentinvention will employ, unless otherwise indicated, conventional methodsof synthetic organic chemistry, mass spectroscopy, preparative andanalytical methods of chromatography, protein chemistry, biochemistry,recombinant DNA techniques and pharmacology. Conventional methods oforganic chemistry include those included in March's Advanced OrganicChemistry: Reactions, 5 Mechanisms, and Structure, 6^(th) Edition, M. B.Smith and J. March, John Wiley & Sons, Inc., Hoboken, NJ, 2007.

The term “alkyl,” either alone or within other terms such as “haloalkyl”and “alkylamino,” embraces linear or branched radicals having one toabout twelve carbon atoms. “Lower alkyl” radicals have one to about sixcarbon atoms. Examples of such radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,hexyl and the like. The term “alkylene” embraces bridging divalentlinear and branched alkyl radicals. Examples include methylene,ethylene, propylene, isopropylene and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twelve carbon atoms.“Lower alkenyl” radicals having two to about six carbon atoms. Examplesof alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyland 4-methylbutenyl. The terms “alkenyl” and “lower alkenyl,” embraceradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations.

The term “alkynyl” denotes linear or branched radicals having at leastone carbon-carbon triple bond and having two to about twelve carbonatoms. “Lower alkynyl” radicals having two to about six carbon atoms.Examples of such radicals include propargyl, butynyl, and the like.

Alkyl, alkenyl, and alkynyl radicals may be optionally substituted withone or more functional groups such as halo, hydroxy, nitro, amino,cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.

The term “alkylamino” embraces “N-alkylamino” and “N,N-dialkylamino”where amino groups are independently substituted with one alkyl radicaland with two alkyl radicals, respectively. “Lower alkylamino” radicalshave one or two alkyl radicals of one to six carbon atoms attached to anitrogen atom. Suitable alkylamino radicals may be mono or dialkylaminosuch as N-methylamino, N-ethylamino, N.N-dimethylamino, N,N-diethylaminoand the like.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms.

The term “haloalkyl” embraces radicals wherein any one or more of thealkyl carbon atoms is substituted with one or more halo as definedabove. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkylradicals including perhaloalkyl. A monohaloalkyl radical, for oneexample, may have an iodo, bromo, chloro or fluoro atom within theradical. Dihalo and polyhaloalkyl radicals may have two or more of thesame halo atoms or a combination of different halo radicals. “Lowerhaloalkyl” embraces radicals having 1-6 carbon atoms. Examples ofhaloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Perfluoroalkyl” means an alkyl radical having allhydrogen atoms replaced with fluoro atoms. Examples includetrifluoromethyl and pentafluoroethyl.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one or two rings wherein such rings may be attachedtogether in a fused manner. The term “aryl” embraces aromatic radicalssuch as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl.

More preferred aryl is phenyl. Said “aryl” group may have 1 or moresubstituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro,cyano, alkoxy, lower alkylamino, and the like. An aryl group may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “heterocyclyl” (or “heterocyclo”) embraces saturated, andpartially saturated heteroatom-containing ring radicals, where theheteroatoms may be selected from nitrogen, sulfur and oxygen.Heterocyclic rings comprise monocyclic 6-8 membered rings, as well as5-16 membered bicyclic ring systems (which can include bridged fused andspiro-fused bicyclic ring systems). It does not include rings containing—O—O—. —O—S— or —S—S— portions. Said “heterocyclyl” group may have 1 to3 substituents such as hydroxyl, Boc, halo, haloalkyl, cyano, loweralkyl, lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, andthe like.

Examples of saturated heterocyclo groups include saturated 3- to6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms[e.g. pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,piperazinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,thiazolidinyl]. Examples of partially saturated heterocyclyl radicalsinclude dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl,and the like.

Particular examples of partially saturated and saturated heterocyclogroups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl,thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl,indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl,isochromanyl, chromanyl, 1,2-dihydroquinolyl,1,2,3,4-tetrahydro-isoquinolyl, 1,2,3,4-tetrahydro-quinolyl,2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl,5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl,3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl,2,3-dihydro-1H-1λ′-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryland dihydrothiazolyl, and the like.

Heterocyclo groups also includes radicals where heterocyclic radicalsare fused/condensed with aryl radicals: unsaturated condensedheterocyclic group containing 1 to 5 nitrogen atoms, for example,indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo[1,5-b]pyridazinyl]; unsaturated condensed heterocyclic group containing1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. benzoxazolyl,benzoxadiazolyl]; unsaturated condensed heterocyclic group containing 1to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., benzothiazolyl,benzothiadiazolyl]; and saturated, partially unsaturated and unsaturatedcondensed heterocyclic group containing 1 to 2 oxygen or sulfur atoms[e.g. benzofuryl, benzothienyl, 2,3-dihydro-benzo[1,4]dioxinyl anddihydrobenzofuryl].

The term “heteroaryl” denotes aryl ring systems that contain one or moreheteroatoms selected from the group O, N and S, wherein the ringnitrogen and sulfur atom(s) are optionally oxidized, and nitrogenatom(s) are optionally quarternized. Examples include unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g.,4H-1,2,4-triazolyl, IH-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated5- to 6-membered heteromonocyclic group containing an oxygen atom, forexample, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-memberedheteromonocyclic group containing a sulfur atom, for example, 2-thienyl,3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example,oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl]; unsaturated 5 to 6-memberedheteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g.,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl].

The term “heteroarylalkyl” denotes alkyl radicals substituted with aheteroaryl group. Examples include pyridylmethyl and thienylethyl.

The term “sulfonyl”, whether used alone or linked to other terms such asalkylsulfonyl, denotes respectively divalent radicals —SO₂—.

The terms “carboxy” or “carboxyl”, whether used alone or with otherterms, such as “carboxyalkyl”, denotes —C(O)—OH.

The term “carbonyl”, whether used alone or with other terms, such as“aminocarbonyl”, denotes —C(O)—.

The term “aminocarbonyl” denotes an amide group of the Formula—C(O)—NH₂.

The terms “heterocycloalkyl” embrace heterocyclic-substituted alkylradicals. Examples include piperidylmethyl and morpholinylethyl.

The term “arylalkyl” embraces aryl-substituted alkyl radicals. Examplesinclude benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkylmay be additionally substituted with halo, alkyl, alkoxy, halkoalkyl andhaloalkoxy.

The term “cycloalkyl” includes saturated carbocyclic groups of 3 to 10carbons. Lower cycloalkyl groups include C₃-C₆ rings. Examples includecyclopentyl, cyclopropyl, and cyclohexyl. Cycloalkyl groups may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “cycloalkylalkyl” embraces cycloalkyl-substituted alkylradicals. “Lower cycloalkylalkyl” radicals are cycloalkyl radicalsattached to alkyl radicals having one to six carbon atoms. Examples ofinclude cyclohexylmethyl. The cycloalkyl in said radicals may beadditionally substituted with halo, alkyl, alkoxy and hydroxy.

The term “cycloalkenyl” includes carbocyclic groups having one or morecarbon-carbon double bonds including “cycloalkyldienyl” compounds.Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl andcycloheptadienyl.

The term “comprising” is meant to be open ended, including the indicatedcomponent but not excluding other elements.

The term “oxo” as used herein contemplates an oxygen atom attached witha double bond.

The term “nitro” as used herein contemplates —NO₂.

The term “cyano” as used herein contemplates —CN.

As used herein, the term “prodrug” means a compound which whenadministered to a host in vivo is converted into the parent drug. Asused herein, the term “parent drug” means any of the presently describedchemical compounds that are useful to treat any of the disordersdescribed herein, or to control or improve the underlying cause orsymptoms associated with any physiological or pathological disorderdescribed herein in a host, typically a human. Prodrugs can be used toachieve any desired effect, including to enhance properties of theparent drug or to improve the pharmaceutic or pharmacokinetic propertiesof the parent. Prodrug strategies exist which provide choices inmodulating the conditions for in vivo generation of the parent drug, allof which are deemed included herein. Nonlimiting examples of prodrugstrategies include covalent attachment of removable groups, or removableportions of groups, for example, but not limited to acylation,phosphorylation, phosphonylation, phosphoramidate derivatives,amidation, reduction, oxidation, esterification, alkylation, othercarboxy derivatives, sulfoxy or sulfone derivatives, carbonylation oranhydride, among others.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist, unless otherwise noted.

In some embodiments, a CDK4/6-replication dependent healthy cell is ahematopoietic stem progenitor cell. Hematopoietic stem and progenitorcells include, but are not limited to, long term hematopoietic stemcells (LT-HSCs), short term hematopoietic stem cells (ST-HSCs),multipotent progenitors (MPPs), common myeloid progenitors (CMPs),common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors(GMPs), and megakaryocyte-erythroid progenitors (MEPs). In someembodiments, the CDK4/6-replication dependent healthy cell may be a cellin a non-hematopoietic tissue, such as, but not limited to, the liver,kidney, pancreas, brain, lung, adrenals, intestine, gut, stomach, skin,auditory system, bone, bladder, ovaries, uterus, testicles, gallbladder,thyroid, heart, pancreatic islets, blood vessels, and the like. In someembodiments, the CDK4/6-replication dependent healthy cell is a renalcell, and in particular a renal epithelial cell, for example, a renalproximal tubule epithelial cells. In some embodiments, aCDK4/6-replication dependent healthy cell is a hematopoietic stemprogenitor cell. In some embodiments, the CDK4/6-replication dependenthealthy cell may be a cell in a non-hematopoietic tissue, such as, butnot limited to, the liver, kidney, pancreas, brain, lung, adrenals,intestine, gut, stomach, skin, auditory system, bone, bladder, ovaries,uterus, testicles, gallbladder, thyroid, heart, pancreatic islets, bloodvessels, and the like.

The term “selective CDK4/6 inhibitor” used in the context of thecompounds described herein includes compounds that inhibit CDK4activity, CDK6 activity, or both CDK4 and CDK6 activity at an IC₅₀ molarconcentration at least about 500, or 1000, or 1500, or 1800, 2000, 5000or 10,000 times less than the IC₅₀ molar concentration necessary toinhibit to the same degree of CDK2 activity in a standardphosphorylation assay.

By “induces G1-arrest” is meant that the inhibitor compound induces aquiescent state in a substantial portion of a cell population at the G1phase of the cell cycle.

By “hematological deficiency” is meant reduced hematological celllineage counts or the insufficient production of blood cells (i.e.,myelodysplasia) and/or lymphocytes (i.e., lymphopenia, the reduction inthe number of circulating lymphocytes, such as B- and T-cells).Hematological deficiency can be observed, for example, asmyelosuppression in form of anemia, reduction in platelet count (i.e.,thrombocytopenia), reduction in white blood cell count (i.e.,leukopenia), or the reduction in granulocytes (e.g., neutropenia).

By “synchronous reentry into the cell cycle” is meant thatCDK4/6-replication dependent healthy cells, for example HSPCs, inG1-arrest due to the effect of a CDK4/6 inhibitor compound reenter thecell-cycle within relatively the same collective timeframe or atrelatively the same rate upon dissipation of the compound's effect.Comparatively, by “asynchronous reentry into the cell cycle” is meantthat the healthy cells, for example HSPCs, in G1 arrest due to theeffect of a CDK4/6 inhibitor compound within relatively differentcollective timeframes or at relatively different rates upon dissipationof the compound's effect such as PD0332991.

By “off-cycle” or “drug holiday” is meant a time period during which thesubject is not administered or exposed to a chemotherapeutic. Forexample, in a treatment regime wherein the subject is administered thechemotherapeutic for 21 straight days and is not administered thechemotherapeutic for 7 days, and the regime is repeated a number oftimes, the 7 day period of non-administration is considered the“off-cycle” or “drug holiday.” Off-target and drug holiday may alsorefer to an interruption in a treatment regime wherein the subject isnot administered the chemotherapeutic for a time due to a deleteriousside effect, for example, myelosuppression.

The subject treated is typically a human subject, although it is to beunderstood the methods described herein are effective with respect toother animals, such as mammals and vertebrate species. Moreparticularly, the term subject can include animals used in assays suchas those used in preclinical testing including but not limited to mice,rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine(pigs and hogs), ruminants, equine, poultry, felines, bovines, murines,canines, and the like.

By “substantial portion” or “significant portion” is meant at least 80%.In alternative embodiments, the portion may be at least 85%, 90% or 95%or greater.

In some embodiments, the term “CDK4/6-replication independent cancer”refers to a cancer that does not significantly require the activity ofCDK4/6 for replication. Cancers of such type are often, but not always,characterized by (e.g., that has cells that exhibit) an increased levelof CDK2 activity or by reduced expression of retinoblastoma tumorsuppressor protein or retinoblastoma family member protein(s), such as,but not limited to p107 and p130. The increased level of CDK2 activityor reduced or deficient expression of retinoblastoma tumor suppressorprotein or retinoblastoma family member protein(s) can be increased orreduced, for example, compared to normal cells. In some embodiments, theincreased level of CDK2 activity can be associated with (e.g., canresult from or be observed along with) MYC proto-oncogene amplificationor overexpression. In some embodiments, the increased level of CDK2activity can be associated with overexpression of Cyclin E1, Cyclin E2,or Cyclin A.

As used herein the term “chemotherapy” or “chemotherapeutic agent”refers to treatment with a cytostatic or cytotoxic agent (i.e., acompound) to reduce or eliminate the growth or proliferation ofundesirable cells, for example cancer cells. Thus, as used herein,“chemotherapy” or “chemotherapeutic agent” refers to a cytotoxic orcytostatic agent used to treat a proliferative disorder, for examplecancer. The cytotoxic effect of the agent can be, but is not required tobe, the result of one or more of nucleic acid intercalation or binding,DNA or RNA alkylation, inhibition of RNA or DNA synthesis, theinhibition of another nucleic acid-related activity (e.g., proteinsynthesis), or any other cytotoxic effect.

Thus, a “cytotoxic agent” can be any one or any combination of compoundsalso described as “antineoplastic” agents or “chemotherapeutic agents.”Such compounds include, but are not limited to, DNA damaging compoundsand other chemicals that can kill cells. “DNA damaging chemotherapeuticagents” include, but are not limited to, alkylating agents, DNAintercalators, protein synthesis inhibitors, inhibitors of DNA or RNAsynthesis, DNA base analogs, topoisomerase inhibitors, and telomeraseinhibitors or telomeric DNA binding compounds. For example, alkylatingagents include alkyl sulfonates, such as busulfan, improsulfan, andpiposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa,and uredepa; ethylenimines and methylmelamines, such as altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; and nitroso ureas, such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimustine.

Antibiotics used in the treatment of cancer include dactinomycin,daunorubicin, doxorubicin, idarubicin, bleomycin sulfate, mytomycin,plicamycin, and streptozocin. Chemotherapeutic antimetabolites includemercaptopurine, thioguanine, cladribine, fludarabine phosphate,fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate,and azathioprine, acyclovir, adenine β-1-D-arabinoside, amethopterin,aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine,6-azauracil, 2′-azido-2′-deoxynucleosides, 5-bromodeoxycytidine,cytosine β-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, and hydroxyurea.

Chemotherapeutic protein synthesis inhibitors include abrin,aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide,diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride,5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate andguanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, andO-methyl threonine. Additional protein synthesis inhibitors includemodeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin,ricin, shiga toxin, showdomycin, sparsomycin, spectinomycin,streptomycin, tetracycline, thiostrepton, and trimethoprim. Inhibitorsof DNA synthesis, include alkylating agents such as dimethyl sulfate,mitomycin C, nitrogen and sulfur mustards; intercalating agents, such asacridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene,ethidium bromide, propidium diiodide-intertwining; and other agents,such as distamycin and netropsin. Topoisomerase inhibitors, such ascoumermycin, nalidixic acid, novobiocin, and oxolinic acid; inhibitorsof cell division, including colcemide, colchicine, vinblastine, andvincristine; and RNA synthesis inhibitors including actinomycin D,α-amanitine and other fungal amatoxins, cordycepin (3′-deoxyadenosine),dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin, andstreptolydigin also can be used as the DNA damaging compound.

Current chemotherapeutic agents whose toxic effects can be mitigated bythe presently disclosed selective CDK4/6 inhibitors include, but are notlimited to, adrimycin, 5-fluorouracil (5FU), 6-mercaptopurine,gemcitabine, melphalan, chlorambucil, mitomycin, irinotecan,mitoxantrone, etoposide, camptothecin, actinomycin-D, mitomycin,cisplatin, hydrogen peroxide, carboplatin, procarbazine,mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, tamoxifen, taxol, transplatinum, vinblastine,vinblastin, carmustine, cytarabine, mechlorethamine, chlorambucil,streptozocin, lomustine, temozolomide, thiotepa, altretamine,oxaliplatin, campothecin, and methotrexate, and the like, and similaracting-type agents. In one embodiment, the DNA damaging chemotherapeuticagent is selected from the group consisting of cisplatin, carboplatin,campothecin, doxorubicin, and etoposide.

In certain alternative embodiments, the CDK4/6 inhibitors describedherein are used for an anti-cancer or anti-proliferative effect incombination with a chemotherapeutic to treat a CDK4/6 replicationindependent, such as an Rb-negative, cancer or proliferative disorder.The CDK4/6 inhibitors described herein may provide an additive orsynergistic effect to the chemotherapeutic, resulting in a greateranti-cancer effect than seen with the use of the chemotherapeutic alone.In one embodiment, the CDK4/6 inhibitors described herein can becombined with one or more of the chemotherapeutic compounds describedabove. In one embodiment, a CDK4/6 inhibitor described herein can becombined with a chemotherapeutic selected from, but not limited to, butnot limited to, tamoxifen, midazolam, letrozole, bortezomib,anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitors, dualmTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors,HSP inhibitors (for example, HSP70 and HSP 90 inhibitors, or acombination thereof), BCL-2 inhibitors, apopototic inducing compounds,AKT inhibitors, including but not limited to, MK-2206, GSK690693,Perifosine, (KRX-0401), GDC-0068, Triciribine, AZD5363, Honokiol,PF-04691502, and Miltefosine, PD-1 inhibitors including but not limitedto, Nivolumab, CT-011, MK-3475, BMS936558, and AMP-514 or FLT-3inhibitors, including but not limited to, P406, Dovitinib, Quizartinib(AC220), Amuvatinib (MP-470), Tandutinib (MLN518), ENMD-2076, andKW-2449, or combinations thereof. Examples of mTOR inhibitors includebut are not limited to rapamycin and its analogs, everolimus (Afinitor),temsirolimus, ridaforolimus, sirolimus, and deforolimus. Examples of P13kinase inhibitors include but are not limited to Wortmannin,demethoxyviridin, perifosine, idelalisib, PX-866, IPI-145 (Infinity),BAY 80-6946, BEZ235, RP6503, TGR 1202 (RP5264), MLN1117 (INK1117),Pictilisib, Buparlisib, SAR245408 (XL147), SAR245409 (XL765), Palomid529, ZSTK474, PWT33597, RP6530, CUDC-907, and AEZS-136. Examples of MEKinhibitors include but are not limited to Tametinib, Selumetinib,MEK162, GDC-0973 (XL518), and PD0325901. Examples of RAS inhibitorsinclude but are not limited to Reolysin and siG12D LODER. Examples ofALK inhibitors include but are not limited to Crizotinib, AP26113, andLDK378. HSP inhibitors include but are not limited to Geldanamycin or17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol. In oneembodiment, the CDK4/6 inhibitor combined with the chemotherapeutic isselected from the group consisting of a compound or compositioncomprising Formula I, Formula II, Formula III, Formula IV, or Formula Vdescribed above, or a pharmaceutically acceptable composition, salt,isotopic analog or prodrug thereof. In one embodiment, the compound isselected from the compounds provided for in Table 1, or apharmaceutically acceptable composition, salt, isotopic analog orprodrug thereof. In one embodiment, the compound is selected fromcompounds T, Q, GG, U, or AAAA, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof.

In one embodiment, a CDK4/6 inhibitor described herein can be combinedwith a chemotherapeutic selected from, but are not limited to, Imatinibmesylate (Gleevac®), Dasatinib (Sprycel®), Nilotinib (Tasigna®),Bosutinib (Bosulif®), Trastuzumab (Herceptin®), Pertuzumab (Perjeta™),Lapatinib (Tykerb®), Gefitinib (Iressa®), Erlotinib (Tarceva®),Cetuximab (Erbitux®), Panitumumab (Vectibix®), Vandetanib (Caprelsa®),Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), Romidepsin (Istodax®),Bexarotene (Tagretin®), Alitretinoin (Panretin®), Tretinoin (Vesanoid®),Carfilizomib (Kyprolis™), Pralatrexate (Folotyn®), Bevacizumab(Avastin®), Ziv-aflibercept (Zaltrap®), Sorafenib (Nexavar®), Sunitinib(Sutent®), Pazopanib (Votrient®), Regorafenib (Stivarga®), andCabozantinib (Cometriq™).

By “long-term hematological toxicity” is meant hematological toxicityaffecting a subject for a period lasting more than one or more weeks,months, or years following administration of a chemotherapeutic agent.Long-term hematological toxicity can result in bone marrow disordersthat can cause the ineffective production of blood cells (i.e.,myelodysplasia) and/or lymphocytes (i.e., lymphopenia, the reduction inthe number of circulating lymphocytes, such as B- and T-cells).Hematological toxicity can be observed, for example, as anemia,reduction in platelet count (i.e., thrombocytopenia) or reduction inwhite blood cell count (i.e., neutropenia). In some cases,myelodysplasia can result in the development of leukemia. Long-termtoxicity related to chemotherapeutic agents can also damage otherself-renewing cells in a subject, in addition to hematological cells.Thus, long-term toxicity can also lead to graying and frailty.

Active Compounds

In one embodiment, the invention is directed to compounds or the use ofsuch compounds of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof,

-   -   wherein:    -   Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)—        wherein z is 2, 3 or 4;    -   each X is independently CH or N;    -   each X′ is independently, CH or N;    -   X″ is independently CH₂, S or NH, arranged such that the moiety        is a stable 5-membered ring;    -   R, R⁸, and R¹¹ are independently H, C₁-C₃ alkyl or haloalkyl,        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O or S; -(alkylene)_(m)-C₃-C₈ cycloalkyl,        -(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which may be optionally independently substituted with        one or more R groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atoms may optionally        combine to form a ring;    -   each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl,        wherein each of said alkyl, cycloalkyl and haloalkyl groups        optionally includes O or N heteroatoms in place of a carbon in        the chain and two R¹'s on adjacent ring atoms or on the same        ring atom together with the ring atom(s) to which they are        attached optionally form a 3-8-membered cycle;    -   y is 0, 1, 2, 3 or 4;    -   R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,        -(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴;        -(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring and wherein m is 0 or 1 and n is 0, 1 or        2;    -   R³ and R⁴ at each occurrence are independently:        -   (i) hydrogen or        -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,            cycloalkylalkyl, heterocycloalkyl, arylalkyl, or            heteroarylalkyl any of which may be optionally independently            substituted with one or more R^(x) groups as allowed by            valance, and wherein two R^(x) groups bound to the same or            adjacent atom may optionally combine to form a ring; or R³            and R⁴ together with the nitrogen atom to which they are            attached may combine to form a heterocyclo ring optionally            independently substituted with one or more R^(x) groups as            allowed by valance, and wherein two R^(x) groups bound to            the same or adjacent atom may optionally combine to form a            ring;    -   R⁵ and R⁵* at each occurrence is:        -   (i) hydrogen or        -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,            heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or            heteroarylalkyl any of which may be optionally independently            substituted with one or more R^(x) groups as allowed by            valance;    -   R^(x) at each occurrence is independently, halo, cyano, nitro,        oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,        -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,        -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,        -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,        -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,        -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(O)—OR⁵)-(alkylene)_(m)-N(R³)—C(S)—OR⁵,        or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:        -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,            cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,            heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl            groups may be further independently substituted with one or            more -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵*,            -(alkylene)_(m)-S(O)_(n)—R⁵*, -(alkylene)_(m)-NR³*R⁴*,            -(alkylene)_(m)-C(O)—R⁵*,            -(alkylene)_(m)-C(═S)R⁵*-(alkylene)_(m)-C(═O)O R⁵*,            -(alkylene)_(m)-OC(═O)R⁵*, -(alkylene)_(m)-C(S)—OR⁵*,            -(alkylene)_(m)-C(O)—NR³*R⁴*, -(alkylene)_(m)-C(S)—NR³*R⁴*,            -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,            -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,            -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,            -(alkylene)_(m)-N(R³*)—C(S)—R⁵*,            -(alkylene)_(m)-O—C(O)—NR³*R⁴*,            -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-SO₂—NR³*R⁴*,            -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,            -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,            -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*-(alkylene)_(m)-N(R³*)—C(S)—OR⁵*,            or -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -   n is 0, 1 or 2, and        -   m is 0 or 1;    -   R³* and R⁴* at each occurrence are independently:        -   (i) hydrogen or        -   (ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl,            heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or            heteroarylalkyl any of which may be optionally independently            substituted with one or more R^(x) groups as allowed by            valance; or R³* and R⁴* together with the nitrogen atom to            which they are attached may combine to form a heterocyclo            ring optionally independently substituted with one or more            R^(x) groups as allowed by valance; and    -   R⁶ is H or lower alkyl, -(alkylene)_(m)-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atoms may optionally        combine to form a ring; and    -   R¹⁰ is (i) NHR^(A), wherein R^(A) is unsubstituted or        substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O, and S; TT is an unsubstituted or substituted        C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl,        unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted        or substituted C₁-C₆ alkylamino, unsubstituted or substituted        di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl,        unsubstituted or substituted heteroaryl comprising one or two 5-        or 6-member rings and 1-4 heteroatoms selected from N, O and S,        unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted        or substituted heterocycle comprising one or two 5- or 6-member        rings and 1-4 heteroatoms selected from N, O and S; or (ii)        —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³        is R^(A);        or a pharmaceutically acceptable salt, prodrug or isotopic        variant, for example, partially or fully deuterated form        thereof.

In some aspects, the compound is of Formula I or Formula II and R⁶ isabsent.

In some aspects, the compound is of Formula III:

and the variables are as defined for compounds of Formulae I and II andpharmaceutically acceptable salts thereof.

In some aspects, R^(x) is not further substituted.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2.

In some aspects, R⁸ is hydrogen or C₁-C₃ alkyl.

In some aspects, R is hydrogen or C₁-C₃ alkyl.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ any of which may beoptionally independently substituted with one or more R^(x) groups asallowed by valance, and wherein two R^(x) groups bound to the same oradjacent atom may optionally combine to form a ring.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ without furthersubstitution.

In some aspects, m in R² is 1. In a further aspect, the alkylene in R²is methylene.

In some aspects, R² is

wherein:

-   -   R²* is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-,        -(alkylene)_(m)-S(O)₂-(alkylene)_(m)- and        -(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is        independently 0 or 1;    -   P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl        group;    -   each R^(x1) is independently        -(alkylene)_(m)-(C(O))_(m)-(alkylene)_(m)-(N(R^(N)))_(m)-(alkyl)_(m)        wherein each m is independently 0 or 1 provided at least one m        is 1, —(C(O))—O-alkyl, -(alkylene)_(m)-cycloalkyl wherein m is 0        or 1, —N(R^(N))-cycloalkyl, —C(O)-cycloalkyl,        -(alkylene)_(m)-heterocyclyl wherein m is 0 or 1, or        —N(R^(N))-heterocyclyl, —C(O)-heterocyclyl,        —S(O)₂-(alkylene)_(m) wherein m is 1 or 2, wherein:        -   R^(N) is H, C₁ to C₄ alkyl or C₁ to C₆ heteroalkyl, and        -   wherein two R^(x1) can, together with the atoms to which            they attach on P, which may be the same atom, form a ring;            and    -   t is 0, 1 or 2.

In some aspects, each R^(x1) is only optionally substituted byunsubstituted alkyl, halogen or hydroxy.

In some aspects, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl.

In some aspects, at least one R^(x1) is -(alkylene)_(m)-heterocyclylwherein m is 0 or 1.

In some aspects, R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some aspects, R² is

In some aspects, R² is

In some aspects, R² is

wherein:

-   -   R²* is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-,        -(alkylene)_(m)-S(O)₂-(alkylene)_(m)- and        -(alkylene)_(m)-NH-(alkylene)_(m)- wherein each m is        independently 0 or 1;    -   P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl        group;    -   P1 is a 4- to 6-membered monocyclic saturated heterocyclyl        group;    -   each R² is independently hydrogen or alkyl; and    -   s is a 0, 1 or 2.

In some aspects, R² is

In some aspects, P1 includes at least one nitrogen.

In some aspects, any alkylene in R²* in any previous aspect is notfurther substituted.

In some aspects, R² is selected from the structures depicted in FIGS.24-26 .

In some aspects, R² is

In some aspects, the compound has general Formula I and morespecifically one of the general structures in FIGS. 27-31 wherein thevariables are as previously defined.

In some aspects, the compound has general Formula Ia:

wherein R¹, R², R and y are as previously defined.

In some embodiments, the compound has Formula Ia and R is alkyl.

In some embodiments, the compound has Formula Ia and R is H.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl andR²* is as previously defined.

In some embodiments, the compound has Formula Ib:

wherein R² and R are as previously defined.

In some embodiments, the compound has Formula Ib and R is alkyl.

In some embodiments, the compound has Formula Ib and R is H.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ic:

wherein R² and R are as previously defined.

In some embodiments, the compound has Formula Ic and R is alkyl.

In some embodiments, the compound has Formula Ic and R is H.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Id:

wherein R² and R are as previously defined.

In some embodiments, the compound has Formula Id and R is alkyl.

In some embodiments, the compound has Formula Id and R is H.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ie:

In some embodiments, the compound has Formula Ie and R is alkyl.

In some embodiments, the compound has Formula Ie and R is H.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula If.

In some embodiments, the compound has Formula If and R is alkyl.

In some embodiments, the compound has Formula If and R is H.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ig:

In some embodiments, the compound has Formula Ig and R is alkyl.

In some embodiments, the compound has Formula Ig and R is H.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ih:

In some embodiments, the compound has Formula Ih and R is alkyl.

In some embodiments, the compound has Formula Ih and R is H.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ii:

In some embodiments, the compound has Formula Ii and R is alkyl.

In some embodiments, the compound has Formula Ii and R is H.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ij:

In some embodiments, the compound has Formula Ij and R is alkyl.

In some embodiments, the compound has Formula Ij and R is H.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Ij and R is H, and both Xare N.

In some embodiments, the compound has the structure:

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Il:

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Im:

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIa:

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIb:

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some aspects, the active compound is:

In certain embodiments, the compound is selected from:

-   -   wherein R is C(H)X, NX, C(H)Y, or C(X)₂,    -   where X is straight, branched or cyclic C₁ to C₅ alkyl group,        including methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl,        sec-butyl, tert-butyl, isobutyl, cyclobutyl, pentyl, isopentyl,        neopentyl, tert-pentyl, sec-pentyl, and cyclopentyl; and    -   Y is NR₁R₂ wherein R₁ and R₂ are independently X, or wherein R₁        and R₂ are alkyl groups that together form a bridge that        includes one or two heteroatoms (N, O, or S);    -   And wherein two X groups can together form an alkyl bridge or a        bridge that includes one or two heteroatoms (N, S, or O) to form        a spiro compound.

The IUPAC name for Formula T is2′-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one;for Formula Q is2′-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one;for Formula GG is2′-((5-(4-isopropylpiperazin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one;and for Formula U is2′-((5-(4-morpholinopiperidin-1-yl)pyridin-2-yl)amino)-7′,8′-dihydro-6′H-spiro[cyclohexane-1,9′-pyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyrimidin]-6′-one.

Further specific compounds that fall within the present invention andthat can be used in the disclosed methods of treatment and compositionsinclude the structures listed in Table 1 below.

TABLE 1 Structures of CDK4/6 Inhibitors Structure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

QQQ

RRR

SSS

TTT

UUU

VVV

WWW

XXX

Isotopic Substitution

The present invention includes compounds and the use of compounds withdesired isotopic substitutions of atoms, at amounts above the naturalabundance of the isotope, i.e., enriched. Isotopes are atoms having thesame atomic number but different mass numbers, i.e., the same number ofprotons but a different number of neutrons. By way of general exampleand without limitation, isotopes of hydrogen, for example, deuterium(²H) and tritium (H) may be used anywhere in described structures.Alternatively or in addition, isotopes of carbon, e.g., ¹³C and ¹⁴C, maybe used. A preferred isotopic substitution is deuterium for hydrogen atone or more locations on the molecule to improve the performance of thedrug. The deuterium can be bound in a location of bond breakage duringmetabolism (an α-deuterium kinetic isotope effect) or next to or nearthe site of bond breakage (a β-deuterium kinetic isotope effect).

Substitution with isotopes such as deuterium can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Substitution of deuterium for hydrogen at a site ofmetabolic break down can reduce the rate of or eliminate the metabolismat that bond. At any position of the compound that a hydrogen atom maybe present, the hydrogen atom can be any isotope of hydrogen, includingprotium (H), deuterium (²H) and tritium (H). Thus, reference herein to acompound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

The term “isotopically-labeled” analog refers to an analog that is a“deuterated analog”, a “¹³C-labeled analog,” or a“deuterated/¹³C-labeled analog.” The term “deuterated analog” means acompound described herein, whereby a H-isotope, i.e., hydrogen/protium(H), is substituted by a H-isotope, i.e., deuterium (²H). Deuteriumsubstitution can be partial or complete. Partial deuterium substitutionmeans that at least one hydrogen is substituted by at least onedeuterium. In certain embodiments, the isotope is 90, 95 or 99% or moreenriched in an isotope at any location of interest. In some embodimentsit is deuterium that is 90, 95 or 99% enriched at a desired location.

CDK-Replication Dependent Cells and Cyclin-Dependent Kinase Inhibitors

Tissue-specific stem cells and subsets of other resident proliferatingcells are capable of self-renewal, meaning that they are capable ofreplacing themselves throughout the adult mammalian lifespan throughregulated replication. Additionally, stem cells divide asymmetrically toproduce “progeny” or “progenitor” cells that in turn produce variouscomponents of a given organ. For example, in the hematopoietic system,the hematopoietic stem cells give rise to progenitor cells which in turngive rise to all the differentiated components of blood (e.g., whiteblood cells, red blood cells, and platelets). See FIG. 1 .

Certain proliferating cells, such as HSPCs, require the enzymaticactivity of the proliferative kinases cyclin-dependent kinase 4 (CDK4)and/or cyclin-dependent kinase 6 (CDK6) for cellular replication. Incontrast, the majority of proliferating cells in adult mammals (e.g.,the more differentiated blood-forming cells in the bone marrow) do notrequire the activity of CDK4 and/or CDK6 (i.e., CDK4/6). Thesedifferentiated cells can proliferate in the absence of CDK4/6 activityby using other proliferative kinases, such as cyclin-dependent kinase 2(CDK2) or cyclin-dependent kinase 1 (CDK1).

The CDK4/6 inhibitor administered is selected from the group consistingof a compound or composition comprising Formula I, Formula II, FormulaIII, Formula IV, or Formula V, or a combination thereof. In oneembodiment, the compound is selected from the compounds described inTable 1.

In certain embodiments, the CDK4/6 inhibitor is a CDK4/6 inhibitor ofFormula I, II, III, IV, or V or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof, wherein theprotection afforded by the compound is short term and transient innature, allowing a significant portion of the cells to synchronouslyrenter the cell-cycle quickly following the cessation of thechemotherapeutic agent's effect, for example within less than about 24,30, 36, or 40 hours. In one embodiment, the compound is selected fromthe compounds described in Table 1 or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof. In oneembodiment, the compound is selected from compounds T, Q, GG, U, orAAAA, or a pharmaceutically acceptable composition, salt, isotopicanalog or prodrug thereof. Cells that are quiescent within the G1 phaseof the cell cycle are more resistant to the damaging effect ofchemotherapeutic agents than proliferating cells. CDK4/6 inhibitorycompounds for use in the described methods are highly selective, potentCDK4/6 inhibitors, with minimal CDK2 inhibitory activity. In oneembodiment, a CDK4/6 compound for use in the methods described hereinhas a CDK4/CycD1 IC₅₀ inhibitory concentration value that is >1500times, >1800 times, >2000 times, >2200 times, >2500 times, >2700times, >3000 times, >3200 times or greater lower than its respectiveIC₅₀ concentration value for CDK2/CycE inhibition. In one embodiment, aCDK4/6 inhibitor for use in the methods described herein has an IC₅₀concentration value for CDK4/CycD1 inhibition that is about <1.50 nM,<1.25 nM, <1.0 nM, <0.90 nM, <0.85 nM, <0.80 nM, <0.75 nM, <0.70 nM,<0.65 nM, <0.60 nM, <0.55 nM, or less. In one embodiment, a CDK4/6inhibitor for use in the methods described herein has an IC₅₀concentration value for CDK2/CycE inhibition that is about >1.0μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 PM, >2.75μM, >3.0 μM, >3.25 μM, >3.5 μM or greater. In one embodiment, a CDK4/6inhibitor for use in the methods described herein has an IC₅₀concentration value for CDK2/CycA IC₅₀ that is >0.80 μM, >0.85 μM, >0.90μM, >0.95 μM, >0.1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25μM, >2.50 μM, >2.75 uM, >3.0 μM or greater. In one embodiment, theCDK4/6 inhibitor for use in the methods described herein are selectedfrom the group consisting of Formula I, Formula II, Formula III, FormulaIV, or Formula V, or a pharmaceutically acceptable composition, salt, orprodrug, thereof. In one embodiment, the compound is selected from thecompounds described in Table 1, or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof. In oneembodiment, the compound is selected from compounds T, Q, GG, U, orAAAA, or a pharmaceutically acceptable composition, salt, isotopicanalog or prodrug thereof.

In one embodiment, the CDK4/6 inhibitors described herein are used inCDK4/6-replication dependent healthy cell cycling strategies wherein asubject is exposed to regular, repeated chemotherapeutic treatments,wherein the healthy cells are G1-arrested when chemotherapeutic agentexposed and allowed to reenter the cell-cycle before the subject's nextchemotherapeutic treatment. Such cycling allows CDK4/6-replicationdependent cells to regenerate damaged blood cell lineages betweenregular, repeated treatments, for example those associated with standardchemotherapeutic treatments for cancer, and reduces the risk associatedwith long term CDK4/6 inhibition. This cycling between a state ofG1-arrest and a state of replication is not feasible in limitedtime-spaced, repeated chemotherapeutic agent exposures using longeracting CDK4/6 inhibitors such as PD0332991, as the lingeringG1-arresting effects of the compound prohibit significant and meaningfulreentry into the cell-cycle before the next chemotherapeutic agentexposure or delay the healthy cells from entering the cell cycle andreconstituting damaged tissues or cells following treatment cessation.

Proliferative disorders that are treated with chemotherapy includecancerous and non-cancer diseases. In a typical embodiment, theproliferative disorder is a CDK4/6-replication independent disorder. Thecompounds are effective in protecting healthy CDK4/6-replicationdependent cells, for example HSPCs, during chemotherapeutic treatment ofa broad range of tumor types, including but not limited to thefollowing: breast, prostate, ovarian, skin, lung, colorectal, brain(i.e., glioma) and renal. Preferably, the selective CDK4/6 inhibitorshould not compromise the efficacy of the chemotherapeutic agent orarrest G1 arrest the cancer cells. Many cancers do not depend on theactivities of CDK4/6 for proliferation as they can use the proliferativekinases promiscuously (e.g., can use CDK 1/2/4/or 6) or lack thefunction of the retinoblastoma tumor suppressor protein (Rb), which isinactivated by the CDKs. The potential sensitivity of certain tumors toCDK4/6 inhibition can be deduced based on tumor type and moleculargenetics using standard techniques. Cancers that are not typicallyaffected by the inhibition of CDK4/6 are those that can be characterizedby one or more of the group including, but not limited to, increasedactivity of CDK1 or CDK2, loss, deficiency, or absence of retinoblastomatumor suppressor protein (Rb), high levels of MYC expression, increasedcyclin E (e.g., E1 or E2) and increased cyclin A, or expression of aRb-inactivating protein (such as HPV-encoded E7). Such cancers caninclude, but are not limited to, small cell lung cancer, retinoblastoma,HPV positive malignancies like cervical cancer and certain head and neckcancers, MYC amplified tumors such as Burkitts' Lymphoma, and triplenegative breast cancer; certain classes of sarcoma, certain classes ofnon-small cell lung carcinoma, certain classes of melanoma, certainclasses of pancreatic cancer, certain classes of leukemia, certainclasses of lymphoma, certain classes of brain cancer, certain classes ofcolon cancer, certain classes of prostate cancer, certain classes ofovarian cancer, certain classes of uterine cancer, certain classes ofthyroid and other endocrine tissue cancers, certain classes of salivarycancers, certain classes of thymic carcinomas, certain classes of kidneycancers, certain classes of bladder cancers, and certain classes oftesticular cancers.

The loss or absence of retinoblastoma (Rb) tumor suppressor protein(Rb-null) can be determined through any of the standard assays known toone of ordinary skill in the art, including but not limited to WesternBlot, ELISA (enzyme linked immunoadsorbent assay), IHC(immunohistochemistry), and FACS (fluorescent activated cell sorting).The selection of the assay will depend upon the tissue, cell line orsurrogate tissue sample that is utilized e.g., for example Western Blotand ELISA may be used with any or all types of tissues, cell lines orsurrogate tissues, whereas the IHC method would be more appropriatewherein the tissue utilized in the methods of the present invention wasa tumor biopsy. FACs analysis would be most applicable to samples thatwere single cell suspensions such as cell lines and isolated peripheralblood mononuclear cells. See for example, US 20070212736 “FunctionalImmunohistochemical Cell Cycle Analysis as a Prognostic Indicator forCancer”.

Alternatively, molecular genetic testing may be used for determinationof retinoblastoma gene status. Molecular genetic testing forretinoblastoma includes the following as described in Lohmann and Gallie“Retinoblastoma. Gene Reviews” (2010)http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=retinoblastomaor Parsam et al. “A comprehensive, sensitive and economical approach forthe detection of mutations in the RB1 gene in retinoblastoma” Journal ofGenetics, 88(4), 517-527 (2009).

Increased activity of CDK1 or CDK2, high levels of MYC expression,increased cyclin E and increased cyclin A can be determined through anyof the standard assays known to one of ordinary skill in the art,including but not limited to Western Blot, ELISA (enzyme linkedimmunoadsorbent assay), IHC (immunohistochemistry), and FACS(fluorescent activated cell sorting). The selection of the assay willdepend upon the tissue, cell line, or surrogate tissue sample that isutilized e.g., for example Western Blot and ELISA may be used with anyor all types of tissues, cell lines, or surrogate tissues, whereas theIHC method would be more appropriate wherein the tissue utilized in themethods of the present invention was a tumor biopsy. FACs analysis wouldbe most applicable to samples that were single cell suspensions such ascell lines and isolated peripheral blood mononuclear cells.

In some embodiments, the cancer is selected from a small cell lungcancer, retinoblastoma, and triple negative (ER/PR/Her2 negative) or“basal-like” breast cancer, which almost always inactivate theretinoblastoma tumor suppressor protein (Rb), and therefore do notrequire CDK4/6 activity to proliferate. Triple negative (basal-like)breast cancer is also almost always genetically or functionally Rb-null.Also, certain virally induced cancers (e.g. cervical cancer and subsetsof Head and Neck cancer) express a viral protein (E7) which inactivatesRb making these tumors functionally Rb-null. Some lung cancers are alsobelieved to be caused by HPV. In one particular embodiment, the canceris small cell lung cancer, and the patient is treated with aDNA-damaging agent selected from the group consisting of etoposide,carboplatin, and cisplatin, or a combination thereof.

The selected CDK4/6 inhibitors described herein can also be used inprotecting healthy CDK4/6-replication dependent cells duringchemotherapeutic treatments of abnormal tissues in non-cancerproliferative diseases, including but not limited to: psoriasis, lupus,arthritis (notably rheumatoid arthritis), hemangiomatosis in infants,multiple sclerosis, myelodegenerative disease, neurofibromatosis,ganglioneuromatosis, keloid formation, Paget's Disease of the bone,fibrocystic disease of the breast, Peyronie's and Duputren's fibrosis,restenosis, and cirrhosis. Further, selective CDK4/6 inhibitors can beused to ameliorate the effects of chemotherapeutic agents in the eventof accidental exposure or overdose (e.g., methotrexate overdose).

According to the present invention, the active compound can beadministered to a subject on any chemotherapeutic treatment schedule andin any dose consistent with the prescribed course of treatment. Theselective CDK4/6 inhibitor compound is administered prior to, during, orfollowing the administration of the chemotherapeutic agent. In oneembodiment, the CDK4/6 inhibitors described herein can be administeredto the subject during the time period ranging from 24 hours prior tochemotherapeutic treatment until 24 hours following exposure. This timeperiod, however, can be extended to time earlier that 24 hour prior toexposure to the agent (e.g., based upon the time it takes thechemotherapeutic agent used to achieve suitable plasma concentrationsand/or the compound's plasma half-life). Further, the time period can beextended longer than 24 hours following exposure to the chemotherapeuticagent so long as later administration of the CDK4/6 inhibitor leads toat least some protective effect. Such post-exposure treatment can beespecially useful in cases of accidental exposure or overdose.

In some embodiments, the selective CDK4/6 inhibitor can be administeredto the subject at a time period prior to the administration of thechemotherapeutic agent, so that plasma levels of the selective CDK4/6inhibitor are peaking at the time of administration of thechemotherapeutic agent. If convenient, the selective CDK4/6 inhibitorcan be administered at the same time as the chemotherapeutic agent, inorder to simplify the treatment regimen. In some embodiments, thechemoprotectant and chemotherapeutic can be provided in a singleformulation.

In some embodiments, the selective CDK4/6 inhibitor can be administeredto the subject such that the chemotherapeutic agent can be administeredeither at higher doses (increased chemotherapeutic dose intensity) ormore frequently (increased chemotherapeutic dose density). Dose-densechemotherapy is a chemotherapy treatment plan in which drugs are givenwith less time between treatments than in a standard chemotherapytreatment plan. Chemotherapy dose intensity represents unit dose ofchemotherapy administered per unit time. Dose intensity can be increasedor decreased through altering dose administered, time interval ofadministration, or both. Myelosuppression continues to represent themajor dose-limiting toxicity of cancer chemotherapy, resulting inconsiderable morbidity and mortality along with frequent reductions inchemotherapy dose intensity, which may compromise disease control andsurvival. The compounds and their use as described herein represent away of increasing chemotherapy dose density and/or dose intensity whilemitigating adverse events such as, but not limited to, myelosuppression.

If desired, multiple doses of the selected CDK4/6 inhibitor compound canbe administered to the subject. Alternatively, the subject can be givena single dose of the selected CDK4/6 inhibitor. For example, theCDK4/6-inhibitor can be administered so that CDK4/6-replicationdependent healthy cells are G1 arrested during chemotherapeutic agentexposure wherein, due to the rapid dissipation of the G1-arrestingeffect of the compounds, a significant number of healthy cells reenterthe cell-cycle and are capable of replicating shortly afterchemotherapeutic agent exposure, for example, within about 24-48 hoursor less, and continue to replicate until administration of theCDK4/6-inhibitor in anticipation of the next chemotherapeutic treatment.In one embodiment, the CDK4/6-inhibitor is administered to allow for thecycling of the CDK4/6-replication dependent healthy cells betweenG1-arrest and reentry into the cell-cycle to accommodate arepeated-dosing chemotherapeutic treatment regimen, for example,including but not limited to a treatment regimen wherein thechemotherapeutic agent is administered: on day 1-3 every 21 days; ondays 1-3 every 28 days; on day 1 every 3 weeks; on day 1, day 8, and day15 every 28 days, on day 1 and day 8 every 28 days; on days 1 and 8every 21 days; on days 1-5 every 21 days; 1 day a week for 6-8 weeks; ondays 1, 22, and 43; days 1 and 2 weekly; days 1-4 and 22-25; 1-4; 22-25,and 43-46; and similar type-regimens, wherein the CDK4/6-replicationdependent cells are G1 arrested during chemotherapeutic agent exposureand a significant portion of the cells reenter the cell-cycle in betweenchemotherapeutic agent exposure.

In one embodiment, the CDK4/6 inhibitor described herein is used toprovide chemoprotection to a subject's CDK4/6-replication dependenthealthy cells during a CDK4/6-replication independent small cell lungcancer treatment protocol. In one embodiment, the CDK4/6 inhibitor isadministered to provide chemoprotection in a small cell lung cancertherapy protocol such as, but not limited to: cisplatin 60 mg/m2 IV onday 1 plus etoposide 120 mg/m2 IV on days 1-3 every 21 d for 4 cycles;cisplatin 80 mg/m2 IV on day 1 plus etoposide 100 mg/m2 IV on days 1-3every 28 d for 4 cycles; cisplatin 60-80 mg/m2 IV on day 1 plusetoposide 80-120 mg/m2 IV on days 1-3 every 21-28 d (maximum of 4cycles); carboplatin AUC 5-6 IV on day 1 plus etoposide 80-100 mg/m2 IVon days 1-3 every 28 d (maximum of 4 cycles); Cisplatin 60-80 mg/m2 IVon day 1 plus etoposide 80-120 mg/m2 IV on days 1-3 every 21-28 d;carboplatin AUC 5-6 IV on day 1 plus etoposide 80-100 mg/m2 IV on days1-3 every 28 d (maximum 6 cycles); cisplatin 60 mg/m2 IV on day 1 plusirinotecan 60 mg/m2 IV on days 1, 8, and 15 every 28 d (maximum 6cycles); cisplatin 30 mg/m2 IV on days 1 and 8 or 80 mg/m2 IV on day 1plus irinotecan 65 mg/m2 IV on days 1 and 8 every 21 d (maximum 6cycles); carboplatin AUC 5 IV on day 1 plus irinotecan 50 mg/m2 IV ondays 1, 8, and 15 every 28 d (maximum 6 cycles); carboplatin AUC 4-5 IVon day 1 plus irinotecan 150-200 mg/m2 IV on day 1 every 21 d (maximum 6cycles); cyclophosphamide 800-1000 mg/m2 IV on day 1 plus doxorubicin40-50 mg/m2 IV on day 1 plus vincristine 1-1.4 mg/m2 IV on day 1 every21-28 d (maximum 6 cycles); Etoposide 50 mg/m2 PO daily for 3 wk every 4wk; topotecan 2.3 mg/m2 PO on days 1-5 every 21 d; topotecan 1.5 mg/m2IV on days 1-5 every 21 d; carboplatin AUC 5 IV on day 1 plus irinotecan50 mg/m2 IV on days 1, 8, and 15 every 28 d; carboplatin AUC 4-5 IV onday 1 plus irinotecan 150-200 mg/m2 IV on day 1 every 21 d; cisplatin 30mg/m2 IV on days 1, 8, and 15 plus irinotecan 60 mg/m2 IV on days 1, 8,and 15 every 28 d; cisplatin 60 mg/m2 IV on day 1 plus irinotecan 60mg/m2 IV on days 1, 8, and 15 every 28 d; cisplatin 30 mg/m2 IV on days1 and 8 or 80 mg/m2 IV on day 1 plus irinotecan 65 mg/m2 IV on days 1and 8 every 21 d; paclitaxel 80 mg/m2 IV weekly for 6 wk every 8 wk;paclitaxel 175 mg/m2 IV on day 1 every 3 wk; etoposide 50 mg/m2 PO dailyfor 3 wk every 4 wk; topotecan 2.3 mg/m2 PO on days 1-5 every 21 d;topotecan 1.5 mg/m2 IV on days 1-5 every 21 d; carboplatin AUC 5 IV onday 1 plus irinotecan 50 mg/m2 IV on days 1, 8, and 15 every 28 d;carboplatin AUC 4-5 IV on day 1 plus irinotecan 150-200 mg/m2 IV on day1 every 21 d; cisplatin 30 mg/m2 IV on days 1, 8, and 15 plus irinotecan60 mg/m2 IV on days 1, 8, and 15 every 28 d; cisplatin 60 mg/m2 IV onday 1 plus irinotecan 60 mg/m2 IV on days 1, 8, and 15 every 28 d;cisplatin 30 mg/m2 IV on days 1 and 8 or 80 mg/m2 IV on day 1 plusirinotecan 65 mg/m2 IV on days 1 and 8 every 21 d; paclitaxel 80 mg/m2IV weekly for 6 wk every 8 wk; and paclitaxel 175 mg/m2 IV on day 1every 3 wk.

In one embodiment, a CDK4/6 inhibitor described herein is administeredto a subject with small cell lung cancer on days 1, 2, and 3 of atreatment protocol wherein the DNA damaging agent selected from thegroup consisting of carboplatin, etoposide, and cisplatin, or acombination thereof, is administered on days 1, 2, and 3 every 21 days.

In one embodiment, a CDK4/6 inhibitor described herein is used toprovide chemoprotection to a subject's CDK4/6-replication dependenthealthy cells during a CDK4/6-replication independent head and neckcancer treatment protocol. In one embodiment, the CDK4/6 inhibitor isadministered to provide chemoprotection in a CDK4/6-replicationindependent head and neck cancer therapy protocol such as, but notlimited to: cisplatin 100 mg/m2 IV on days 1, 22, and 43 or 40-50 mg/m2IV weekly for 6-7 wk; cetuximab 400 mg/m2 IV loading dose 1 wk beforethe start of radiation therapy, then 250 mg/m2 weekly (premedicate withdexamethasone, diphenhydramine, and ranitidine); cisplatin 20 mg/m2 IVon day 2 weekly for up to 7 wk plus paclitaxel 30 mg/m2 IV on day 1weekly for up to 7 wk; cisplatin 20 mg/m2/day IV on days 1-4 and 22-25plus 5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 and22-25; 5-FU 800 mg/m2 by continuous IV infusion on days 1-5 given on thedays of radiation plus hydroxyurea 1 g PO q12h (11 doses per cycle);chemotherapy and radiation given every other week for a total of 13 wk;carboplatin 70 mg/m2/day IV on days 1-4, 22-25, and 43-46 plus 5-FU 600mg/m2/day by continuous IV infusion on days 1-4, 22-25, and 43-46;carboplatin AUC 1.5 IV on day 1 weekly plus paclitaxel 45 mg/m2 IV onday 1 weekly; cisplatin 100 mg/m2 IV on days 1, 22, and 43 or 40-50mg/m2 IV weekly for 6-7 wk; docetaxel 75 mg/m2 IV on day 1 pluscisplatin 100 mg/m2 IV on day 1 plus 5-FU 100 mg/m2/day by continuous IVinfusion on days 1-4 every 3 wk for 3 cycles, then 3-8 wk later,carboplatin AUC 1.5 IV weekly for up to 7 wk during radiation therapy;docetaxel 75 mg/m2 IV on day 1 plus cisplatin 75 mg/m2 IV on day 1 plus5-FU 750 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk for4 cycles; cisplatin 100 mg/m2 IV on day 1 every 3 wk for 6 cycles plus5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk for6 cycles plus cetuximab 400 mg/m2 IV loading dose on day 1, then 250mg/m2 IV weekly until disease progression (premedicate withdexamethasone, diphenhydramine, and ranitidine); carboplatin AUC 5 IV onday 1 every 3 wk for 6 cycles plus 5-FU 1000 mg/m2/day by continuous IVinfusion on days 1-4 every 3 wk for 6 cycles plus cetuximab 400 mg/m2 IVloading dose on day 1, then 250 mg/m2 IV weekly until diseaseprogression (premedicate with dexamethasone, diphenhydramine, andranitidine); cisplatin 75 mg/m2 IV on day 1 plus docetaxel 75 mg/m2 IVon day 1 every 3 wk; cisplatin 75 mg/m2 IV on day 1 plus paclitaxel 175mg/m2 IV on day 1 every 3 wk; carboplatin AUC 6 IV on day 1 plusdocetaxel 65 mg/m2 IV on day 1 every 3 wk; carboplatin AUC 6 IV on day 1plus paclitaxel 200 mg/m2 IV on day 1 every 3 wk; cisplatin 75-100 mg/m2IV on day 1 every 3-4 wk plus cetuximab 400 mg/m2 IV loading dose on day1, then 250 mg/m2 IV weekly (premedicate with dexamethasone,diphenhydramine, and ranitidine); cisplatin 100 mg/m2 IV on day 1 plus5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk;methotrexate 40 mg/m2 IV weekly (3 wk equals 1 cycle); paclitaxel 200mg/m2 IV every 3 wk; docetaxel 75 mg/m2 IV every 3 wk; cetuximab 400mg/m2 IV loading dose on day 1, then 250 mg/m2 IV weekly until diseaseprogression (premedicate with dexamethasone, diphenhydramine, andranitidine); cisplatin 100 mg/m2 IV on day 1 every 3 wk for 6 cyclesplus 5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3wk for 6 cycles plus cetuximab 400 mg/m2 IV loading dose on day 1, then250 mg/m2 IV weekly (premedicate with dexamethasone, diphenhydramine,and ranitidine); carboplatin AUC 5 IV on day 1 every 3 wk for 6 cyclesplus 5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3wk for 6 cycles plus cetuximab 400 mg/m2 IV loading dose on day 1, then250 mg/m2 IV weekly (premedicate with dexamethasone, diphenhydramine,and ranitidine); cisplatin 75 mg/m2 IV on day 1 plus docetaxel 75 mg/m2IV on day 1 every 3 wk; cisplatin 75 mg/m2 IV on day 1 plus paclitaxel175 mg/m2 IV on day 1 every 3 wk; carboplatin AUC 6 IV on day 1 plusdocetaxel 65 mg/m2 IV on day 1 every 3 wk; carboplatin AUC 6 IV on day 1plus paclitaxel 200 mg/m2 IV on day 1 every 3 wk; cisplatin 75-100 mg/m2IV on day 1 every 3-4 wk plus cetuximab 400 mg/m2 IV loading dose on day1, then 250 mg/m2 IV weekly (premedicate with dexamethasone,diphenhydramine, and ranitidine); cisplatin 100 mg/m2 IV on day 1 plus5-FU 1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk;methotrexate 40 mg/m2 IV weekly (3 wk equals 1 cycle); paclitaxel 200mg/m2 IV every 3 wk; docetaxel 75 mg/m2 IV every 3 wk; cetuximab 400mg/m2 IV loading dose on day 1, then 250 mg/m2 IV weekly until diseaseprogression (premedicate with dexamethasone, diphenhydramine, andranitidine); cisplatin 100 mg/m2 IV on days 1, 22, and 43 withradiation, then cisplatin 80 mg/m2 IV on day 1 plus 5-FU 1000 mg/m2/dayby continuous IV infusion on days 1-4 every 4 wk for 3 cycles; cisplatin75 mg/m2 IV on day 1 plus docetaxel 75 mg/m2 IV on day 1 every 3 wk;cisplatin 75 mg/m2 IV on day 1 plus paclitaxel 175 mg/m2 IV on day 1every 3 wk; carboplatin AUC 6 IV on day 1 plus docetaxel 65 mg/m2 IV onday 1 every 3 wk; carboplatin AUC 6 IV on day 1 plus paclitaxel 200mg/m2 IV on day 1 every 3 wk; cisplatin 100 mg/m2 IV on day 1 plus 5-FU1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk;cisplatin 50-70 mg/m2 IV on day 1 plus gemcitabine 1000 mg/m2 IV on days1, 8, and 15 every 4 wk; gemcitabine 1000 mg/m2 IV on days 1, 8, and 15every 4 wk or gemcitabine 1250 mg/m2 IV on days 1 and 8 every 3 wk;methotrexate 40 mg/m2 IV weekly (3 wk equals 1 cycle); paclitaxel 200mg/m2 IV every 3 wk; docetaxel 75 mg/m2 IV every 3 wk; cisplatin 75mg/m2 IV on day 1 plus docetaxel 75 mg/m2 IV on day 1 every 3 wk;cisplatin 75 mg/m2 IV on day 1 plus paclitaxel 175 mg/m2 IV on day 1every 3 wk; carboplatin AUC 6 IV on day 1 plus docetaxel 65 mg/m2 IV onday 1 every 3 wk; carboplatin AUC 6 IV on day 1 plus paclitaxel 200mg/m2 IV on day 1 every 3 wk; cisplatin 100 mg/m2 IV on day 1 plus 5-FU1000 mg/m2/day by continuous IV infusion on days 1-4 every 3 wk;cisplatin 50-70 mg/m2 IV on day 1 plus gemcitabine 1000 mg/m2 IV on days1, 8, and 15 every 4 wk; gemcitabine 1000 mg/m2 IV on days 1, 8, and 15every 4 wk or gemcitabine 1250 mg/m2 IV on days 1 and 8 every 3 wk;methotrexate 40 mg/m2 IV weekly (3 wk equals 1 cycle); paclitaxel 200mg/m2 IV every 3 wk; and docetaxel 75 mg/m2 IV every 3 wk.

In one embodiment, the CDK4/6 inhibitor described herein is used toprovide chemoprotection to a subject's CDK4/6-replication dependenthealthy cells during a CDK4/6-replication independent triple negativebreast cancer treatment protocol. In one embodiment, the CDK4/6inhibitor is administered to provide chemoprotection in aCDK4/6-replication independent triple negative breast cancer therapyprotocol such as, but not limited to: dose-dense doxorubicin(adriamycin) and cyclophosphamide (cytoxan) every two weeks for fourcycles followed by dose-dense paclitaxel (Taxol) every two weeks forfour cycles; adriamycin/paclitaxel/cyclophosphomide every three weeksfor a total of four cycles; adriamycin/paclitaxel/cyclophosphomide everytwo weeks for a total of four cycles; adriamycin/cyclophosphomidefollowed by paclitaxel (Taxol) every three weeks for four cycles each;and adriamycin/cyclophosphomide followed by paclitaxel (Taxol) every twoweeks for four cycles each.

Triple-negative breast cancer (TNBC) is defined as the absence ofstaining for estrogen receptor, progesterone receptor, and HER2/neu.TNBC is insensitive to some of the most effective therapies availablefor breast cancer treatment including HER2-directed therapy such astrastuzumab and endocrine therapies such as tamoxifen or the aromataseinhibitors. Combination cytotoxic chemotherapy administered in adose-dense or metronomic schedule remains the standard therapy forearly-stage TNBC. Platinum agents have recently emerged as drugs ofinterest for the treatment of TNBC with carboplatin added to paclitaxeland adriamycin plus cyclophosphamide chemotherapy in the neoadjuvantsetting. The poly (ADP-ribose) polymerase (PARP) inhibitors are emergingas promising therapeutics for the treatment of TNBC. PARPs are a familyof enzymes involved in multiple cellular processes, including DNArepair.

As a nonlimiting illustration, the subject is exposed tochemotherapeutic agent at least 5 times a week, at least 4 times a week,at least 3 times a week, at least 2 times a week, at least 1 time aweek, at least 3 times a month, at least 2 times a month, or at least 1time a month, wherein the subject's CDK4/6-replication dependent healthycells are G1 arrested during treatment and allowed to cycle in betweenchemotherapeutic agent exposure, for example during a treatment break.In one embodiment, the subject is undergoing 5 times a weekchemotherapeutic treatment, wherein the subject's CDK4/6-replicationdependent healthy cells are G1 arrested during the chemotherapeuticagent exposure and allowed to reenter the cell-cycle during the 2 daybreak, for example, over the weekend.

In one embodiment, using a CDK4/6 inhibitor described herein, thesubject's CDK4/6-replication dependent healthy cells are arrested duringthe entirety of the chemotherapeutic agent exposure time-period, forexample, during a contiguous multi-day regimens, the cells are arrestedover the time period that is required to complete the contiguousmulti-day course, and then allowed to recycle at the end of thecontiguous multi-day course. In one embodiment, using a CDK4/6 inhibitordescribed herein, the subject's CDK4/6-replication dependent healthycells are arrested during the entirety of the chemotherapeutic regimen,for example, in a daily chemotherapeutic exposure for three weeks, andrapidly reenter the cell-cycle following the completion of thetherapeutic regimen.

In one embodiment, the subject has been exposed to a chemotherapeuticagent, and, using a CDK4/6 inhibitor described herein, the subject'sCDK4/6-replication dependent healthy cells are placed in G1 arrestfollowing exposure in order to mitigate, for example, DNA damage. In oneembodiment, the CDK4/6 inhibitor is administered at least 1%2 hour, atleast 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, atleast 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, atleast 10 hours, at least 12 hours, at least 14 hours, at least 16 hours,at least 18 hours, at least 20 hours or more post chemotherapeutic agentexposure.

In some embodiments, the present invention provides methods forprotection of mammals, particularly humans, from the acute and chronictoxic effects of chemotherapeutic agents by forcing CDK4/6-replicationdependent healthy cells, for example hematopoietic stem and progenitorcells (HSPCs) and/or renal epithelial cells, into a quiescent state bytransient (e.g., over a less than about 40, 36, 30, 24 hour or lessperiod) treatment with a CDK4/6 inhibitor selected from the groupconsisting of Formula I, Formula II, Formula III, Formula IV, or FormulaV or a pharmaceutically acceptable composition, salt, isotopic analog,or prodrug thereof. In one embodiment, the compound is selected from thecompounds described in Table 1 or a pharmaceutically acceptablecomposition, salt, isotopic analog or prodrug thereof. In oneembodiment, the compound is selected from compounds T, Q, GG, U, orAAAA, or a pharmaceutically acceptable composition, salt, isotopicanalog or prodrug thereof. CDK4/6-replication dependent cells recoverfrom this period of transient quiescence, and then function normallyafter treatment with the inhibitor is stopped, and its intra-cellulareffect dissipates. During the period of quiescence, theCDK4/6-replication dependent cells are protected from the effects ofchemotherapeutic agents.

In some embodiments, the CDK4/6-replication dependent healthy cells canbe arrested for longer periods, for example, over a period of hours,days, and/or weeks, through multiple, time separated administrations ofa CDK4/6 inhibitor described herein. Because of the rapid andsynchronous reentry into the cell cycle by CDK4/6-replication dependenthealthy cells, for example HSPCs, upon dissipation of the CDK4/6inhibitors intra-cellular effects, the cells are capable ofreconstituting the cell lineages faster than CDK4/6 inhibitors withlonger G1 arresting profiles, for example PD0332991.

The reduction in chemotoxicity afforded by the selective CDK4/6inhibitors can allow for dose intensification (e.g., more therapy can begiven in a fixed period of time) in medically related chemotherapies,which will translate to better efficacy. Therefore, the presentlydisclosed methods can result in chemotherapy regimens that are lesstoxic and more effective. Also, in contrast to protective treatmentswith exogenous biological growth factors, the selective CDK4/6inhibitors described herein are orally available small molecules, whichcan be formulated for administration via a number of different routes.When appropriate, the small molecules can be formulated for oral,topical, intranasal, inhalation, intravenous or any other desired formof administration.

A CDK4/6 inhibitor useful in the methods described herein is a selectiveCDK4/6 inhibitor compound that selectively inhibit at least one of CDK4and CDK6, or whose predominant mode of action is through inhibition ofCDK4 and/or CDK6. In one embodiment, the selective CDK4/6 inhibitorshave an IC₅₀ for CDK4 as measured in a CDK4/CycD1 IC₅₀ phosphorylationassay that is at least 1500, 2000, 5000 or even 10,000 times or greaterlower than the compound's IC₅₀s for CDK2 as measured in a CDK2/CycE IC₅₀phosphorylation assay. In one embodiment, the CDK4/6 inhibitors are atleast about 10 times or greater more potent (i.e., have an IC₅₀ in aCDK4/CycD1 phosphorylation assay that is at least 10 times or morelower) than PD0332991.

The use of a selected CDK4/6 inhibitor as described herein can induceselective G1 arrest in CDK4/6-dependent cells (e.g., as measured in acell-based in vitro assay). In one embodiment, the CDK4/6 inhibitor iscapable of increasing the percentage of CDK4/6-dependent cells in the G1phase, while decreasing the percentage of CDK4/6-dependent cells in theG2/M phase and S phase. In one embodiment, the selective CDK4/6inhibitor induces substantially pure (i.e., “clean”) G1 cell cyclearrest in the CDK4/6-dependent cells (e.g., wherein treatment with theselective CDK4/6 inhibitor induces cell cycle arrest such that themajority of cells are arrested in G1 as defined by standard methods(e.g. propidium iodide (PI) staining or others) with the population ofcells in the G2/M and S phases combined being less than about 30%, about25%, about 20%, about 15%, about 10%, about 5%, about 3% or less of thetotal cell population. Methods of assessing the cell phase of apopulation of cells are known in the art (see, for example, in U.S.Patent Application Publication No. 2002/0224522) and include cytometricanalysis, microscopic analysis, gradient centrifugation, elutriation,fluorescence techniques including immunofluorescence, and combinationsthereof. Cytometric techniques include exposing the cell to a labelingagent or stain, such as DNA-binding dyes, e.g., PI, and analyzingcellular DNA content by flow cytometry. Immunofluorescence techniquesinclude detection of specific cell cycle indicators such as, forexample, thymidine analogs (e.g., 5-bromo-2-deoxyuridine (BrdU) or aniododeoxyuridine), with fluorescent antibodies.

In some embodiments, the use of a selective CDK4/6 inhibitor describedherein result in reduced or substantially free of off-target effects,particularly related to inhibition of kinases other than CDK4 and orCDK6 such as CDK2, as the selective CDK4/6 inhibitors described hereinare poor inhibitors (e.g., >1 uM IC₅₀) of CDK2. Furthermore, because ofthe high selectivity for CDK4/6, the use of the compounds describedherein should not induce cell cycle arrest in CDK4/6-independent cells.In addition, because of the short transient nature of the G1-arresteffect, the CDK4/6-replication dependent cells more quickly reenter thecell-cycle than, comparatively, use of PD0332991 provides, resulting inthe reduced risk of, in one embodiment, hematological toxicitydevelopment during long term treatment regimens due to the ability ofHSPCs to replicate between chemotherapeutic treatments.

In some embodiments, the use of a selective CDK4/6 inhibitor describedherein reduces the risk of undesirable off-target effects including, butnot limited to, long term toxicity, anti-oxidant effects, and estrogeniceffects. Anti-oxidant effects can be determined by standard assays knownin the art. For example, a compound with no significant anti-oxidanteffects is a compound that does not significantly scavengefree-radicals, such as oxygen radicals. The anti-oxidant effects of acompound can be compared to a compound with known anti-oxidant activity,such as genistein. Thus, a compound with no significant anti-oxidantactivity can be one that has less than about 2, 3, 5, 10, 30, or 100fold anti-oxidant activity relative to genistein. Estrogenic activitiescan also be determined via known assays. For instance, a non-estrogeniccompound is one that does not significantly bind and activate theestrogen receptor. A compound that is substantially free of estrogeniceffects can be one that has less than about 2, 3, 5, 10, 20, or 100 foldestrogenic activity relative to a compound with estrogenic activity,e.g., genistein.

Synthesis of Select CDK4/6 Inhibitors

CDK4/6 Inhibitors of the present invention can be synthesized by anymeans known to those of ordinary skill in the art, including forexample, according to the generalized Schemes of 1 through 9 below.Specific syntheses can be found in, for instance, WO2012/061156(5-(4-isopropylpiperazin-1-yl)pyridine-2-amine and5-(4-morpholino-1-piperidyl)pyridine-2-amine respectively). Formula Iand Formula II can be synthesized according to Scheme 1 using thecorresponding substituted 2-aminopyrimidines or as described inWO2012/061156.

In Scheme 2, Ref-1 is WO 2010/020675 A1; Ref-2 is White, J. D.; et al.J. Org. Chem. 1995, 60, 3600; and Ref-3 Presser, A. and Hufner, A.Monatsheftefur Chemie 2004, 135, 1015.

In Scheme 3, Ref-1 is WO 2010/020675 A1; Ref-4 is WO 2005/040166 A1; andRef-5 is Schoenauer, K and Zbiral, E. Tetrahedron Letters 1983, 24, 573.

In Scheme 4, Ref-1 is WO 2010/020675 A1.

In Scheme 9, Ref-1 is WO 2010/020675 A1; Ref-2 is WO 2005/040166 A1; andRef-3 is Schoenauer, K and Zbiral, E. Tetrahedron Letters 1983, 24, 573.

In one embodiment a lactam intermediate is treated with BOC-anhydride inthe presence of an organic base such as triethylamine in an organicsolvent such as dichloromethane. The Boc protected lactam is treatedwith carbon dioxide in the presence of a nickel catalyst to generate acarboxylic acid. The carboxylic acid is reacted with thionyl chloride inthe presence of an organic solvent such as toluene. The resulting acidchloride is treated with an amine to generate an amide that can bedeprotected with a strong acid such as trifluoroacetic acid to generatethe final target inhibitor compound.

Alternatively, the lactam can be generated by reacting the carboxylicacid with a protected amine in the presence of a strong acid and adehydrating agent, which can be together in one moiety as a strong acidanhydride. Examples of strong acid anhydrides include, but are notlimited to, trifluoroacetic acid anhydride, tribromoacetic acidanhydride, trichloroacetic acid anhydride, or mixed anhydrides. Thedehydrating agent can be a carbodiimide based compound such as but notlimited to DCC (N,N-dicyclohexylcarbodiimide), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or DIC(N,N-diisopropylcarbodiimide). An additional step may be necessary totake off the N-protecting group and the methodologies are known to thoseskilled in the art

Alternatively, the halogen moiety bonded to the pyrimidine ring can besubstituted with any leaving group that can be displaced by a primaryamine, for example to create an intermediate for a final product such asBr, I, F, SMe, SO₂Me, SOalkyl, SO₂alkyl. See, for Example, PCT/US2013/037878 to Tavares.

Other amine intermediates and final amine compounds can be synthesizedby those skilled in the art. It will be appreciated that the chemistrycan employ reagents that comprise reactive functionalities that can beprotected and de-protected and will be known to those skilled in the artat the time of the invention. See for example, Greene, T. W. and Wuts,P. G. M., Greene's Protective Groups in Organic Synthesis, 4^(th)edition, John Wiley and Sons.

Formulas T, Q, GG and U were prepared above were characterized by massspectrometry and NMR as shown below:

Formula T

7.25 (s, 1H) 7.63 (br. s., 2H) 7.94 (br. s., 1H) 8.10 (br. s., 1H) 8.39(br. s., 1H) 9.08 (br. s., 1H) 11.59 (br. s., 1H). LCMS ESI (M+H) 447.

Formula Q

1H NMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d, J=7.32 Hz, 2H) 1.08-1.37 (m,3H) 1.38-1.64 (m, 2H) 1.71 (br. s., 1H) 1.91 (br. s., 1H) 2.80 (br. s.,1H) 3.12 (s, 1H) 3.41 (br. s., 4H) 3.65 (br. s., 4H) 4.09 (br. s., 1H)7.26 (s, 1H) 7.52-7.74 (m, 2H) 7.94 (br. s., 1H) 8.13 (br. s., 1H) 8.40(br. s., 1H) 9.09 (br. s., 1H) 9.62 (br. s., 1H) 11.71 (br. s., 1H).LCMS ESI (M+) 433

Formula GG

1H NMR (600 MHz, DMSO-d₆) δ ppm 0.85 (br. s., 1H) 1.17-1.39 (m, 7H)1.42-1.58 (m, 2H) 1.67-1.84 (m, 3H) 1.88-2.02 (m, 1H) 2.76-2.93 (m, 1H)3.07-3.22 (m, 1H) 3.29-3.39 (m, 1H) 3.41-3.61 (m, 4H) 3.62-3.76 (m, 4H)3.78-3.88 (m, 1H) 4.12 (br. s., 1H) 7.28 (s, 1H) 7.60-7.76 (m, 2H) 7.98(s, 1H) 8.13 (br. s., 1H) 8.41 (s, 1H) 9.10 (br. s., 1H) 11.21 (br. s.,1H) 11.54 (s, 1H). LCMS ESI (M+H) 475

Formula U

1H NMR (600 MHz, DMSO-d₆) δ ppm 0.84 (t, J=7.61 Hz, 2H) 1.13-1.39 (m,4H) 1.46 (d, J=14.05 Hz, 2H) 1.64-1.99 (m, 6H) 2.21 (br. s., 1H)2.66-2.89 (m, 2H) 3.06 (br. s., 1H) 3.24-3.36 (m, 1H) 3.37-3.50 (m, 2H)3.56-3.72 (m, 2H) 3.77-4.00 (m, 4H) 4.02-4.19 (m, 2H) 7.25 (s, 1H)7.50-7.75 (m, 2H) 7.89 (d, J=2.93 Hz, 1H) 8.14 (d, J=7.32 Hz, 1H) 8.38(br. s., 1H) 9.06 (s, 1H) 11.53 (br. s., 1H). LCMS ESI (M+H) 517

Active Compounds, Salts and Formulations

As used herein, the term “active compound” refers to the selective CDK4/6 inhibitor compounds described herein or a pharmaceuticallyacceptable salt or isotopic analog thereof. The active compound can beadministered to the subject through any suitable approach. The amountand timing of active compound administered can, of course, be dependenton the subject being treated, on the dosage of chemotherapy to which thesubject is anticipated of being exposed to, on the time course of thechemotherapeutic agent exposure, on the manner of administration, on thepharmacokinetic properties of the particular active compound, and on thejudgment of the prescribing physician. Thus, because of subject tosubject variability, the dosages given below are a guideline and thephysician can titrate doses of the compound to achieve the treatmentthat the physician considers appropriate for the subject. In consideringthe degree of treatment desired, the physician can balance a variety offactors such as age and weight of the subject, presence of preexistingdisease, as well as presence of other diseases. Pharmaceuticalformulations can be prepared for any desired route of administrationincluding, but not limited to, oral, intravenous, or aerosoladministration, as discussed in greater detail below.

The therapeutically effective dosage of any active compound describedherein will be determined by the health care practitioner depending onthe condition, size and age of the patient as well as the route ofdelivery. In one non-limited embodiment, a dosage from about 0.1 toabout 200 mg/kg has therapeutic efficacy, with all weights beingcalculated based upon the weight of the active compound, including thecases where a salt is employed. In some embodiments, the dosage can bethe amount of compound needed to provide a serum concentration of theactive compound of up to between about 1 and 5, 10, 20, 30, or 40 μM. Insome embodiments, a dosage from about 10 mg/kg to about 50 mg/kg can beemployed for oral administration. Typically, a dosage from about 0.5mg/kg to 5 mg/kg can be employed for intramuscular injection. In someembodiments, dosages can be from about 1 μmol/kg to about 50 μmol/kg,or, optionally, between about 22 μmol/kg and about 33 μmol/kg of thecompound for intravenous or oral administration. An oral dosage form caninclude any appropriate amount of active material, including for examplefrom 5 mg to, 50, 100, 200, or 500 mg per tablet or other solid dosageform.

In accordance with the presently disclosed methods, pharmaceuticallyactive compounds as described herein can be administered orally as asolid or as a liquid, or can be administered intramuscularly,intravenously, or by inhalation as a solution, suspension, or emulsion.In some embodiments, the compounds or salts also can be administered byinhalation, intravenously, or intramuscularly as a liposomal suspension.When administered through inhalation the active compound or salt can bein the form of a plurality of solid particles or droplets having anydesired particle size, and for example, from about 0.01, 0.1 or 0.5 toabout 5, 10, 20 or more microns, and optionally from about 1 to about 2microns. Compounds as disclosed in the present invention havedemonstrated good pharmacokinetic and pharmacodynamics properties, forinstance when administered by the oral or intravenous routes.

In one embodiment of the invention, these improved CDK4/6 inhibitors canbe administered in a concerted regimen with a blood growth factor agent.As such, in one embodiment, the use of the compounds and methodsdescribed herein is combined with the use of hematopoietic growthfactors including, but not limited to, granulocyte colony stimulatingfactor (G-CSF, for example, sold as Neupogen (filgrastin), Neulasta(peg-filgrastin), or lenograstin), granulocyte-macrophage colonystimulating factor (GM-CSF, for example sold as molgramostim andsargramostim (Leukine)), M-CSF (macrophage colony stimulating factor),thrombopoietin (megakaryocyte growth development factor (MGDF), forexample sold as Romiplostim and Eltrombopag) interleukin (IL)-12,interleukin-3, interleukin-11 (adipogenesis inhibiting factor oroprelvekin), SCF (stem cell factor, steel factor, kit-ligand, or KL) anderythropoietin (EPO), and their derivatives (sold as for exampleepoetin-α as Darbopoetin, Epocept, Nanokine, Epofit, Epogin, Eprex andProcrit; epoetin-β sold as for example NeoRecormon, Recormon andMicera), epoetin-delta (sold as for example Dynepo), epoetin-omega (soldas for example Epomax), epoetin zeta (sold as for example Silapo andReacrit) as well as for example Epocept, EPOTrust, Erypro Safe,Repoeitin, Vintor, Epofit, Erykine, Wepox, Espogen, Relipoeitin,Shanpoietin, Zyrop and EPIAO).

The pharmaceutical formulations can comprise an active compounddescribed herein or a pharmaceutically acceptable salt thereof, in anypharmaceutically acceptable carrier. If a solution is desired, water maybe the carrier of choice for water-soluble compounds or salts. Withrespect to the water-soluble compounds or salts, an organic vehicle,such as glycerol, propylene glycol, polyethylene glycol, or mixturesthereof, can be suitable. In the latter instance, the organic vehiclecan contain a substantial amount of water. The solution in eitherinstance can then be sterilized in a suitable manner known to those inthe art, and for illustration by filtration through a 0.22-micronfilter. Subsequent to sterilization, the solution can be dispensed intoappropriate receptacles, such as depyrogenated glass vials. Thedispensing is optionally done by an aseptic method. Sterilized closurescan then be placed on the vials and, if desired, the vial contents canbe lyophilized.

In addition to the active compounds or their salts, the pharmaceuticalformulations can contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the formulations can contain antimicrobialpreservatives. Useful antimicrobial preservatives include methylparaben,propylparaben, and benzyl alcohol. An antimicrobial preservative istypically employed when the formulation is placed in a vial designed formulti-dose use. The pharmaceutical formulations described herein can belyophilized using techniques well known in the art.

For oral administration a pharmaceutical composition can take the formof solutions, suspensions, tablets, pills, capsules, powders, and thelike. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate may be employed along withvarious disintegrants such as starch (e.g., potato or tapioca starch)and certain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia.

Additionally, lubricating agents such as magnesium stearate, sodiumlauryl sulfate, and talc are often very useful for tableting purposes.Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules. Materials in this connection alsoinclude lactose or milk sugar as well as high molecular weightpolyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the compounds of the presentlydisclosed subject matter can be combined with various sweetening agents,flavoring agents, coloring agents, emulsifying agents and/or suspendingagents, as well as such diluents as water, ethanol, propylene glycol,glycerin and various like combinations thereof.

In yet another embodiment of the subject matter described herein, thereis provided an injectable, stable, sterile formulation comprising anactive compound as described herein, or a salt thereof, in a unit dosageform in a sealed container. The compound or salt is provided in the formof a lyophilizate, which is capable of being reconstituted with asuitable pharmaceutically acceptable carrier to form a liquidformulation suitable for injection thereof into a subject. When thecompound or salt is substantially water-insoluble, a sufficient amountof emulsifying agent, which is physiologically acceptable, can beemployed in sufficient quantity to emulsify the compound or salt in anaqueous carrier. Particularly useful emulsifying agents includephosphatidyl cholines and lecithin.

Additional embodiments provided herein include liposomal formulations ofthe active compounds disclosed herein. The technology for formingliposomal suspensions is well known in the art. When the compound is anaqueous-soluble salt, using conventional liposome technology, the samecan be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the active compound, the active compound can besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed can be of any conventionalcomposition and can either contain cholesterol or can becholesterol-free. When the active compound of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced can be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations comprising the active compounds disclosedherein can be lyophilized to produce a lyophilizate, which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. These formulations comprisea solution or suspension of a desired compound described herein or asalt thereof, or a plurality of solid particles of the compound or salt.The desired formulation can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles may for example have a particle size in the range of about 0.5to about 10 microns, and optionally from about 0.5 to about 5 microns.The solid particles can be obtained by processing the solid compound ora salt thereof, in any appropriate manner known in the art, such as bymicronization. Optionally, the size of the solid particles or dropletscan be from about 1 to about 2 microns. In this respect, commercialnebulizers are available to achieve this purpose. The compounds can beadministered via an aerosol suspension of respirable particles in amanner set forth in U.S. Pat. No. 5,628,984, the disclosure of which isincorporated herein by reference in its entirety.

When the pharmaceutical formulation suitable for administration as anaerosol is in the form of a liquid, the formulation can comprise awater-soluble active compound in a carrier that comprises water. Asurfactant can be present, which lowers the surface tension of theformulation sufficiently to result in the formation of droplets withinthe desired size range when subjected to nebulization.

The term “pharmaceutically acceptable salts” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with subjects (e.g., human subjects) withoutundue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the presently disclosed subject matter.

Thus, the term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the presently disclosedsubject matter. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Pharmaceuticallyacceptable base addition salts may be formed with metals or amines, suchas alkali and alkaline earth metal hydroxides, or of organic amines.Examples of metals used as cations, include, but are not limited to,sodium, potassium, magnesium, calcium, and the like. Examples ofsuitable amines include, but are not limited to,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, and procaine.

Salts can be prepared from inorganic acids sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, laurylsulphonate and isethionate salts,and the like. Salts can also be prepared from organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. and the like. Representativesalts include acetate, propionate, caprylate, isobutyrate, oxalate,malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Pharmaceuticallyacceptable salts can include cations based on the alkali and alkalineearth metals, such as sodium, lithium, potassium, calcium, magnesium andthe like, as well as non-toxic ammonium, quaternary ammonium, and aminecations including, but not limited to, ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. Also contemplated are the saltsof amino acids such as arginate, gluconate, galacturonate, and the like.See, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which isincorporated herein by reference.

EXAMPLES

Intermediates B, E, K, L, 1A, 1F and 1CA were synthesized according toU.S. Pat. No. 8,598,186 entitled CDK Inhibitors to Tavares, F. X. andStrum, J. C.

The patents WO 2013/148748 entitled Lactam Kinase Inhibitors to Tavares,F. X., WO 2013/163239 entitled Synthesis of Lactams to Tavares, F. X.,and U.S. Pat. No. 8,598,186 entitled CDK Inhibitors to Tavares, F. X.and Strum, J. C. are incorporated by reference herein in their entirety.

Example 1 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4yl)amino]ethyl]carbamate, Compound 1

To a solution of 5-bromo-2,4-dichloropyrimidine (3.2 g, 0.0135 mol) inethanol (80 mL) was added Hunig's base (3.0 mL) followed by the additionof a solution of N-(tert-butoxycarbonyl)-1,2-diaminoethane (2.5 g,0.0156 mole) in ethanol (20 mL). The contents were stirred overnight for20 hrs. The solvent was evaporated under vacuum. Ethyl acetate (200 mL)and water (100 mL) were added and the layers separated. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum. Column chromatography on silica gel using hexane/ethyl acetate(0-60%) afforded tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. ¹HNMR(d6-DMSO) δ ppm 8.21 (s, 1H), 7.62 (brs, 1H), 7.27 (brs, 1H), 3.39 (m,2H), 3.12 (m, 2H), 1.34 (s, 9H). LCMS (ESI) 351 (M+H).

Example 2 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate,Compound 2

To tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate (1.265 g,3.6 mmol) in THE (10 mL) was added the acetal (0.778 mL, 5.43 mmol),Pd(dppf)CH₂Cl₂ (148 mg), and triethylamine (0.757 mL, 5.43 mmol). Thecontents were degassed and then purged with nitrogen. To this was thenadded CuI (29 mg). The reaction mixture was heated at reflux for 48 hrs.After cooling, the contents were filtered over CELITE™ and concentrated.Column chromatography of the resulting residue using hexane/ethylacetate (0-30%) afforded tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.¹HNMR (d6-DMSO) δ ppm 8.18 (s, 1H), 7.63 (brs, 1H), 7.40 (brs, 1H), 5.55(s, 1H), 3.70 (m, 2H), 3.60 (m, 2H), 3.42 (m, 2H), 3.15 (m, 2H),1.19-1.16 (m, 15H). LCMS (ESI) 399 (M+H).

Example 3 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 3

To a solution of the coupled product (2.1 g, 0.00526 mole) in THE (30mL) was added TBAF solid (7.0 g). The contents were heated to andmaintained at 65 degrees for 2 hrs. Concentration followed by columnchromatography using ethyl acetate/hexane (0-50%) afforded tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale brown liquid (1.1 g). ¹HNMR (d6-DMSO) δ ppm 8.88 (s, 1H), 6.95(brs, 1H), 6.69 (s, 1H), 5.79 (s, 1H), 4.29 (m, 2H), 3.59 (m, 4H), 3.34(m, 1H), 3.18 (m, 1H), 1.19 (m, 9H), 1.17 (m, 6H). LCMS (ESI) 399 (M+H).

Example 4 Synthesis of tert-butylN-[2-(2-chloro-6-formyl-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]carbamate,Compound 4

To the acetal (900 mg) from the preceeding step was added AcOH (8.0 mL)and water (1.0 mL). The reaction was stirred at room temperature for 16hrs. Conc. and column chromatography over silica gel using ethylacetate/hexanes (0-60%) afforded tert-butylN-[2-(2-chloro-6-formyl-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]carbamate asa foam (0.510 g). ¹HNMR (d6-DMSO) δ ppm 9.98 (s, 1H), 9.18 (s, 1H), 7.66(s, 1H), 6.80 (brs, 1H), 4.52 (m, 2H), 4.36 (m, 2H), 1.14 (s, 9H). LCMS(ESI) 325 (M+H).

Example 5 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 5

To the aldehyde (0.940 g) from the preceeding step in DMF (4 mL) wasadded oxone (1.95 g, 1.1 eq). The contents were stirred at room temp for7 hrs. Silica gel column chromatography using hexane/ethyl acetate(0-100%) afforded7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.545 g). ¹HNMR (d6-DMSO) δ ppm 9.11 (s, 1H), 7.39 (s, 1H), 4.38(m, 2H), 4.15 (m, 2H), 1.48 (m, 9H). LCMS (ESI) 341 (M+H).

Example 6 Synthesis of methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylate,Compound 6

To a solution of 2-chloro-7-propyl-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.545 g, 0.00156 mole) from the preceeding step in toluene (3.5mL) and MeOH (1 mL) was added TMS-diazomethane (1.2 mL). After stirringovernight at room temperature, the excess of TMS-diazomethane wasquenched with acetic acid (3 mL) and the reaction was concentrated undervacuum. The residue was purified by silica gel column chromatographywith hexane/ethyl acetate (0-70%) to afford methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylateas an off white solid (0.52 g). ¹HNMR (d6-DMSO) δ ppm 9.10 (s, 1H), 7.45(s, 1H), 6.81 (brs, 1H) 4.60 (m, 2H), 3.91 (s, 3H), 3.29 (m, 2H), 1.18(m, 9H) LCMS (ESI) 355 (M+H).

Example 7 Synthesis of Chloro tricyclic amide, Compound 7

To methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylate(0.50 g, 0.0014 mole) from the preceeding step in dichloromethane (2.0mL) was added TFA (0.830 mL). The contents were stirred at roomtemperature for 1 hr. Concentration under vacuum afforded the crudeamino ester which was suspended in toluene (5 mL) and Hunig's base (0.5mL). The contents were heated at reflux for 2 hrs. Concentrationfollowed by silica gel column chromatography using hexane/ethyl acetate(0-50%) afforded the desired chloro tricyclic amide (0.260 g). ¹HNMR(d6-DMSO) δ ppm 9.08 (s, 1H), 8.48 (brs, 1H), 7.21 (s, 1H) 4.33 (m, 2H),3.64 (m, 2H). LCMS (ESI) 223 (M+H).

Example 8 Synthesis of chloro-N-methyltricyclic amide, Compound 8

To a solution of the chloro tricycliclactam, Compound 7, (185 mg,0.00083 mole) in DMF (2.0 mL) was added sodium hydride (55% dispersionin oil, 52 mg). After stirring for 15 mins, methyl iodide (62 μL, 1.2eq). The contents were stirred at room temperature for 30 mins. Afterthe addition of methanol (5 mL), sat NaHCO₃ was added followed by theaddition of ethyl acetate. Separation of the organic layer followed bydrying with magnesium sulfate and concentration under vacuum affordedthe N-methylated amide in quantitative yield. ¹HNMR (d6-DMSO) δ ppm 9.05(s, 1H), 7.17 (s, 1H) 4.38 (m, 2H), 3.80 (m, 2H), 3.05 (s, 3H). LCMS(ESI) 237 (M+H).

Example 9 Synthesis of 1-methyl-4-(6-nitro-3-pyridyl)piperazine,Compound 9

To 5-bromo-2-nitropyridine (4.93 g, 24.3 mmole) in DMF (20 mL) was addedN-methylpiperazine (2.96 g, 1.1 eq) followed by the addition of DIPEA(4.65 mL, 26.7 mmole). The contents were heated at 90 degrees for 24hrs. After addition of ethyl acetate (200 mL), water (100 mL) was addedand the layers separated. Drying followed by concentration afforded thecrude product which was purified by silica gel column chromatographyusing (0-10%) DCM/Methanol. ¹HNMR (d6-DMSO) δ ppm 8.26 (s, 1H), 8.15(1H, d, J=9.3 Hz), 7.49 (1H, d, J=9.4 Hz), 3.50 (m, 4H), 2.49 (m, 4H),2.22 (s, 3H).

Example 10 Synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine,Compound 10

To 1-methyl-4-(6-nitro-3-pyridyl)piperazine (3.4 g) in ethyl acetate(100 mL) and ethanol (100 mL) was added 10% Pd/C (400 mg) and then thereaction was stirred under hydrogen (10 psi) overnight. After filtrationthrough CELITE™, the solvents were evaporated and the crude product waspurified by silica gel column chromatography using DCM/7N ammonia inMeOH (0-5%) to afford 5-(4-methylpiperazin-1-yl)pyridin-2-amine (2.2 g).T HNMR (d6-DMSO) δ ppm 7.56 (1H, d, J=3 Hz), 7.13 (1H, m), 6.36 (1H, d,J=8.8 Hz), 5.33 (brs, 2H), 2.88 (m, 4H), 2.47 (m, 4H), 2.16 (s, 3H).

Example 11 Synthesis of tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate, Compound 11

This compound was prepared as described in WO 2010/020675 A1.

Example 12 Synthesis of tert-butylN-[2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, Compound 12

To benzyl N-[1-(hydroxymethyl)-2-methyl-propyl]carbamate (11.0 g, 0.0464mole) in dioxane (100 mL) cooled to 0° C. was added diphenylphosphorylazide (10.99 mL, 1.1 eq) followed by the addition of DBU (8.32 mL, 1.2eq). The contents were allowed to warm to room temperature and stirredfor 16 hrs. After the addition of ethyl acetate (300 mL) and water (100mL), the organic layer was separated and washed with satd. NaHCO₃ (100mL). The organic layer was then dried (magnesium sulfate) andconcentrated under vacuum. To this intermediate in DMSO (100 mL) wasadded sodium azide (7.54 g) and the contents then heated to 90 degreesfor 2 hrs. After addition of ethyl acetate and water the layers wereseparated. The organic layer was dried with magnesium sulfate followedby concentration under vacuum to afford an oil that was purified bysilica gel column chromatography using hexane/ethyl acetate (0-70%) toafford benzyl N-[1-(azidomethyl)-2-methyl-propyl] carbamate 6.9 g as acolorless oil.

To benzyl N-[1-(azidomethyl)-2-methyl-propyl] carbamate (6.9 g, 0.0263mole) in THE (100 mL) was added triphenyl phosphine (7.59 g, 1.1 eq).The contents were stirred for 20 hrs. After addition of water (10 mL),and stirring for an additional 6 hrs, ethyl acetate was added and thelayers separated. After drying with magnesium sulfate and concentrationunder vacuum, the crude product was purified by silica gel columnchromatography using DCM/MeOH (0-10%) to afford benzylN-[1-(aminomethyl)-2-methyl-propyl] carbamate as a yellow oil.

To benzyl N-[1-(aminomethyl)-2-methyl-propyl] carbamate (4.65 g, 0.019mole) in THE (70 mL) was added 2N NaOH (20 mL) followed by the additionof di-tert-butyl dicarbonate (5.15 g, 1.2 eq). After stirring for 16hrs, ethyl acetate was added and the layers separated. After drying withmagnesium sulfate and concentration under vacuum, the crude product waspurified using hexane/ethyl acetate (0-40%) over a silica gel column toafford intermediate A, tert-butylN-[2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, (6.1 g). ¹HNMR(600 μMHz, CHLOROFORM-d) δ ppm 0.89 (d, J=6.73 Hz, 3H) 0.92 (d, J=6.73Hz, 3H) 1.38 (s, 9H) 1.70-1.81 (m, 1H) 3.18 (d, J=5.56 Hz, 2H) 3.47-3.60(m, 1H) 4.76 (s, 1H) 4.89 (d, J=7.90 Hz, 1H) 5.07 (s, 2H) 7.25-7.36 (m,5H). LCMS (ESI) 337 (M+H).

Example 13 Synthesis of tert-butylN-[2-(benzyloxycarbonylamino)-4-methyl-pentyl] carbamate, Compound 13

To a solution of benzyl N-[1-(hydroxymethyl)-3-methyl-butyl]carbamate(6.3 g, 0.025 mole) in DCM (100 mL) was added diisopropylethyl amine(5.25 mL, 1.2 eq) followed by the addition of methane sulfonylchloride(2.13 mL, 1.1 eq) at 0 degrees. After stirring for 3 hrs, water (100 mL)was added and the organic layer separated. After drying with magnesiumsulfate and concentration under vacuum, the crude[2-(benzyloxycarbonylamino)-4-methyl-pentyl]methanesulfonate which wastaken directly to the next step.

To the crude [2-(benzyloxycarbonylamino)-4-methyl-pentyl]methanesulfonate from the above reaction in DMF (50 mL), was addedsodium azide 2.43 g. The reaction mixture was then heated to 85 degreesfor 3 hrs. After cooling, ethyl acetate (300 mL) and water was added.The organic layer was separated, dried with magnesium sulfate and thenconcentrated under vacuum to afford the crude benzylN-[1-(azidomethyl)-3-methyl-butyl] carbamate. To this crude intermediatewas added THE (100 mL) followed by triphenylphosphine 7.21 g and stirredunder nitrogen for 16 hrs. After addition of water (10 mL), and stirringfor an additional 6 hrs, ethyl acetate was added and the layersseparated. After drying with magnesium sulfate and concentration undervacuum, the crude product was columned using DCM/MeOH (0-10%) to affordbenzyl N-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g).

To benzyl N-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g, 0.018mole) in THE (60 mL) was added 2N NaOH (18 mL) followed by the additionof di-tert-butyl dicarbonate (4.19 g, 1.07 eq). After stirring for 16hrs, ethyl acetate was added and the layers separated. After drying withmagnesium sulfate and concentration under vacuum, the crude product wastaken to the next step. ¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.89 (d,J=6.73 Hz, 6H) 1.25-1.34 (m, 1H) 1.39 (s, 9H) 1.57-1.71 (m, 2H)3.04-3.26 (m, 2H) 3.68-3.80 (m, 1H) 4.72-4.89 (m, 2H) 5.06 (s, 2H)7.25-7.38 (m, 5H). LCMS (ESI) 351 (M+H).

Example 14 Synthesis of tert-butylN-[(2R)-2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, Compound14

Compound 14 was synthesized from benzylN-[(1R)-1-(hydroxymethyl)-2-methyl-propyl]carbamate using similarsynthetic steps as that described for Compound 13. The analytical data(NMR and mass spec) was consistent with that for Compound 12.

Example 15 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3-methyl-butyl]carbamate, Compound 15

Compound 15 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2-methyl-propyl]carbamate using similarsynthetic steps as that described for Compound 13. The analytical data(NMR and mass spec) was consistent with that for Compound 12.

Example 16 Synthesis of tert-butylN-[(1S)-1-(aminomethyl)-2-methyl-propyl]carbamate, Compound 16

To a solution of tert-butylN-[(1S)-1-(hydroxymethyl)-2-methyl-propyl]carbamate carbamate (6.3 g,0.025 mole) in THE (100 mL) was added diisopropylethyl amine (5.25 mL,1.2 eq) followed by the addition of methane sulfonylchloride (2.13 mL,1.1 eq) at 0 degrees. After stirring for 3 hrs, water (100 mL) was addedand the organic layer separated. After drying with magnesium sulfate andconcentration under vacuum, the crude[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl] methanesulfonate wastaken directly to the next step.

To the crude [(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]methanesulfonate from the above reaction in DMSO (50 mL), was addedsodium azide (2.43 g). The reaction mixture was then heated to 85degrees for 3 hrs. After cooling, ethyl acetate (300 mL) and water wereadded. The organic layer was separated, dried with magnesium sulfate andthen concentrated under vacuum to afford the crude benzylN-[1-(azidomethyl)-3-methyl-butyl] carbamate. To this crude intermediatewas added THE (100 mL) followed by triphenylphosphine (7.21 g) and thereaction was stirred under nitrogen for 16 hrs. After addition of water(10 mL), and stirring for an additional 6 hrs, ethyl acetate was addedand the layers separated. After drying with magnesium sulfate andconcentration under vacuum, the crude product was purified by silica gelcolumn chromatography using DCM/MeOH (0-10%) to afford benzylN-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g). LCMS (ESI) 203(M+H).

Example 17 Synthesis of tert-butylN-[(1R)-1-(aminomethyl)-2-methyl-propyl]carbamate, Compound 17

Compound 17 was synthesized from tert-butylN-[(1R)-1-(hydroxymethyl)-2-methyl-propyl] carbamate using a similarsynthetic sequence as described for Compound 16. The analytical data(NMR and mass spec) was consistent with Compound 16.

Example 18 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentyl] carbamate, Compound18

Compound 18 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-3-methyl-butyl]carbamate using a similarsynthetic sequence as described for Compound 13. The analytical data(NMR and mass spec) was consistent with Compound 13.

Example 19 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-2-phenyl-ethyl] carbamate, Compound19

Compound 19 was synthesized from benzylN-[(1S)-2-hydroxy-1-phenyl-ethyl] carbamate using a similar syntheticsequence as described for Compound 13. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.20-1.33 (m, 9H) 3.11 (t, J=6.29 Hz, 2H) 4.59-4.68 (m, 1H) 4.88-5.01(m, 2H) 6.81 (t, J=5.42 Hz, 1H) 7.14-7.35 (m, 10H) 7.69 (d, J=8.49 Hz,1H). LCMS (ESI) 371 (M+H).

Example 20 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3-methyl-pentyl] carbamate, Compound20

Compound 20 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2-methyl-butyl]carbamate using a similarsynthetic sequence as described for Compound 13. ¹HNMR (600 MHz,CHLOROFORM-d) δ ppm 0.85-0.92 (m, 6H) 1.05-1.15 (m, 1H) 1.35-1.41 (m,9H) 1.45-1.56 (m, 2H) 3.14-3.24 (m, 2H) 3.54-3.64 (m, 1H) 4.78 (s, 1H)4.96 (d, J=7.91 Hz, 1H) 5.06 (s, 2H) 7.27-7.37 (m, 5H). LCMS (ESI) 351(M+H).

Example 21 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3,3-dimethyl-butyl] carbamate,Compound 21

Compound 21 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2,2-dimethyl-propyl]carbamate using a similarsynthetic sequence as described for Compound 13. LCMS (ESI) 351.

Example 22 Synthesis of tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl] carbamate, Compound 22

To a solution of benzyl N-[1-(aminomethyl)cyclohexyl]carbamate (10.0 g,0.0381 mole) in THE (150 mL) was added di-tert-butyl dicarbonate (9.15g, 1.1 eq) and the contents were stirred at room temperature for 16 hrs.Ethyl acetate and water were then added. The organic layer wasseparated, dried over magnesium sulfate and then concentrated undervacuum to afford tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl] carbamate (13.1 g).¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.92-1.54 (m, 17H) 1.76-2.06 (m, 2H) 3.09(d, J=6.15 Hz, 2H) 4.92 (s, 2H) 6.63 (d, J=17.27 Hz, 1H) 7.16-7.49 (m,6H). LCMS (ESI) 363 (M+H).

Example 23 Synthesis of tert-butylN-[[1-(benzyloxycarbonylamino)cyclopentyl]methyl] carbamate, Compound 23

tert-butyl N-[[1-(benzyloxycarbonylamino)cyclopentyl]methyl]carbamatewas synthesized in an analogous manner to tert-butylN-[[1-(benzyloxycarbonylamino) cyclohexyl]methyl] carbamate. LCMS (ESI)349 (M+H).

Example 24 Synthesis of 2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine,Compound 24

To 5-bromo-2-nitropyridine (1.2 g, 5.9 mmol) in DMSO (4 mL) was added1-(4-piperidyl)piperidine (1.0 g, 5.9 mmole) and triethylamine (0.99 mL,7.1 mmole). The contents were heated to 120° C. in a CEM Discoverymicrowave system for 3 hours. The crude reaction was then purified bysilica gel column chromatography with DCM/methanol (0-20%) to afford2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine as an oil (457 mg).¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.26-1.36 (m, 2H) 1.43 (m, 6H) 1.76 (m,2H) 2.37 (m, 5H) 2.94 (t, J=12.74 Hz, 2H) 4.06 (d, J=13.47 Hz, 2H) 7.41(dd, J=9.37, 2.64 Hz, 1H) 8.08 (d, J=9.37 Hz, 1H) 8.20 (d, J=2.64 Hz,1H).

Example 25 Synthesis of 5-[4-(1-piperidyl)-1-piperidyl]pyridin-2-amine,Compound 25

5-[4-(1-piperidyl)-1-piperidyl]pyridin-2-amine was prepared in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.13-1.37 (m, 6H) 1.40-1.63 (m, 6H) 1.71 (m, 2H), 2.24 (m, 1H) 2.43(m, 2H) 3.33 (d, J=12.30 Hz, 2H) 5.31 (s, 2H) 6.33 (d, J=8.78 Hz, 1H)7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.64 Hz, 1H). LCMS (ESI) 261(M+H).

Example 26 Synthesis of 4-[1-(6-nitro-3-pyridyl)-4-piperidyl]morpholine, Compound 26

4-[1-(6-nitro-3-pyridyl)-4-piperidyl]morpholine was synthesized in amanner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.41 (m, 2H) 1.82 (m, 2H) 2.42 (m, 5H) 2.98 (t, J=12.44Hz, 2H) 3.52 (s, 4H) 4.04 (d, J=12.88 Hz, 2H) 7.42 (d, J=9.37 Hz, 1H)8.08 (d, J=9.08 Hz, 1H) 8.21 (s, 1H).

Example 27 Synthesis of 5-(4-morpholino-1-piperidyl) pyridin-2-amine,Compound 27

5-(4-morpholino-1-piperidyl)pyridin-2-amine was prepared in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.34-1.52 (m, 2H) 1.78 (m, 2H) 2.14 (m, 1H) 2.43 (m, 4H) 3.32 (d,J=12.30 Hz, 4H) 3.47-3.59 (m, 4H) 5.32 (s, 2H) 6.34 (d, J=8.78 Hz, 1H)7.11 (dd, J=8.93, 2.78 Hz, 1H) 7.47-7.62 (m, 1H). LCMS (ESI) 263 (M+H).

Example 28 Synthesis of 4-[1-(6-nitro-3-pyridyl)-4-piperidyl]thiomorpholine, Compound 28

4-[1-(6-nitro-3-pyridyl)-4-piperidyl] thiomorpholine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.40-1.52 (m, 2H) 1.71 (m, 2H) 2.49-2.55 (m, 4H)2.56-2.63 (m, 1H) 2.68-2.75 (m, 4H) 2.88-2.98 (m, 2H) 4.09 (d, J=13.18Hz, 2H) 7.42 (dd, J=9.22, 3.07 Hz, 1H) 8.08 (d, J=9.37 Hz, 1H) 8.20 (d,J=3.22 Hz, 1H).

Example 29 Synthesis of 5-(4-thiomorpholino-1-piperidyl)pyridin-2-amine, Compound 29

5-(4-thiomorpholino-1-piperidyl) pyridin-2-amine was prepared in amanner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆)₆ ppm1.47-1.59 (m, 2H) 1.65 (m, 2H) 2.22-2.38 (m, 1H) 2.50-2.59 (m, 6H)2.68-2.82 (m, 4H) 3.33 (d, J=12.00 Hz, 2H) 5.31 (s, 2H) 6.33 (d, J=9.08Hz, 1H) 7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.64 Hz, 1H). LCMS(ESI) 279 (M+H).

Example 30 Synthesis of 2-nitro-5-(1-piperidyl)pyridine, Compound 30

2-nitro-5-(1-piperidyl) pyridine was synthesized in a manner similar tothat used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.56 (m, 6H) 3.49 (d, J=4.39 Hz, 4H) 7.30-7.47 (m, 1H)8.02-8.12 (m, 1H) 8.15-8.26 (m, 1H).

Example 31 Synthesis of 5-(1-piperidyl)pyridin-2-amine, Compound 31

5-(1-piperidyl) pyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.39-1.46 (m, 2H) 1.51-1.62 (m, 4H)2.75-2.92 (m, 4H) 5.30 (s, 2H) 6.34 (d, J=8.78 Hz, 1H) 7.09 (dd, J=8.78,2.93 Hz, 1H) 7.54 (d, J=2.93 Hz, 1H). LCMS (ESI) 178 (M+H).

Example 32 Synthesis of 4-(6-nitro-3-pyridyl) thiomorpholine, Compound32

4-(6-nitro-3-pyridyl) thiomorpholine was synthesized in a manner similarto that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 2.56-2.69 (m, 4H) 3.79-3.92 (m, 4H) 7.43 (dd, J=9.22,3.07 Hz, 1H) 8.10 (d, J=9.37 Hz, 1H) 8.20 (d, J=2.93 Hz, 1H).

Example 33 Synthesis of 5-thiomorpholinopyridin-2-amine, Compound 33

5-thiomorpholinopyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 2.59-2.73 (m, 4H) 3.04-3.20 (m, 4H) 5.41(s, 2H) 6.35 (d, J=8.78 Hz, 1H) 7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.57 (d,J=2.64 Hz, 1H). LCMS (ESI) 196 (M+H).

Example 34 Synthesis of tert-butyl(4R)-5-(6-nitro-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate,Compound 34

tert-butyl(4R)-5-(6-nitro-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylatewas synthesized in a manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.33 (d, J=32.21 Hz, 11H) 1.91 (m, 2H) 3.15 (d, J=10.25Hz, 1H) 3.58 (m, 1H) 4.46 (m, 1H) 4.83 (s, 1H) 7.16 (s, 1H) 7.94 (s, 1H)8.05-8.16 (m, 1H).

Example 35 Synthesis of tert-butyl(4R)-5-(6-amino-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate,Compound 35

tert-butyl(4R)-5-(6-amino-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylatewas prepared in a manner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.31 (d, J=31.91 Hz, 11H) 1.83 (m, 2H) 2.71-2.82 (m, 1H) 3.44 (m,1H) 4.30 (d, 2H) 5.08 (s, 2H) 6.35 (d, J=8.78 Hz, 1H) 6.77-6.91 (m, 1H)7.33 (s, 1H). LCMS (ESI) 291 (M+H).

Example 36 Synthesis of N,N-dimethyl-1-(6-nitro-3-pyridyl)piperidin-4-amine, Compound 36

N,N-dimethyl-1-(6-nitro-3-pyridyl)piperidin-4-amine was synthesized in amanner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.30-1.45 (m, 2H) 1.79 (m, 2H) 2.14 (s, 6H) 2.33 (m, 1H)2.92-3.04 (m, 2H) 4.03 (d, J=13.76 Hz, 2H) 7.42 (dd, J=9.22, 3.07 Hz,1H) 8.04-8.11 (m, 1H) 8.21 (d, J=2.93 Hz, 1H).

Example 37 Synthesis of 5-[4-(dimethylamino)-1-piperidyl]pyridin-2-amine, Compound 37

5-[4-(dimethylamino)-1-piperidyl]pyridin-2-amine was prepared in amanner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.35-1.50 (m, 2H) 1.69-1.81 (m, 2H) 2.00-2.10 (m, 1H) 2.11-2.22 (s,6H) 3.17-3.36 (m, 4H) 5.19-5.38 (s, 2H) 6.34 (d, J=8.78 Hz, 1H) 7.10(dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.63 Hz, 1H). LCMS (ESI) 221 (M+H).

Example 38 Synthesis of 4-(6-nitro-3-pyridyl) morpholine, Compound 38

4-(6-nitro-3-pyridyl) morpholine was synthesized in a manner similar tothat used in the synthesis of 2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine.

Example 39 Synthesis of 5-morpholinopyridin-2-amine, Compound 39

5-morpholinopyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 2.91-3.00 (m, 4H) 3.76-3.84 (m, 4H)4.19 (br. s., 2H) 6.45 (d, J=8.78 Hz, 1H) 7.12 (dd, J=8.78, 2.93 Hz, 1H)7.72 (d, J=2.93 Hz, 1H).

Example 40 Synthesis of 5-(4-isobutylpiperazin-1-yl) pyridin-2-amine,Compound 40

1-isobutyl-4-(6-nitro-3-pyridyl)piperazine was synthesized in a mannersimilar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then converted5-(4-isobutylpiperazin-1-yl)pyridin-2-amine in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.88 (d, J=6.73 Hz, 6H) 1.71-1.84(m, 1H) 2.10 (d, J=7.32 Hz, 2H) 2.46-2.58 (m, 4H) 2.97-3.07 (m, 4H) 4.12(s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.14 (dd, J=8.78, 2.93 Hz, 1H) 7.75 (d,J=2.93 Hz, 1H). LCMS (ESI) 235 (M+H).

Example 41 Synthesis of 5-(4-isopropylpiperazin-1-yl) pyridin-2-amine,Compound 41

1-isopropyl-4-(6-nitro-3-pyridyl)piperazine was synthesized in a mannersimilar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-(4-isopropylpiperazin-1-yl)pyridin-2-amine in a manner similar tothat used in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 1.06 (d, J=6.44 Hz, 6H) 2.59-2.75(m, 5H) 2.97-3.10 (m, 4H) 4.13 (s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.15 (dd,J=9.08, 2.93 Hz, 1H) 7.76 (d, J=2.93 Hz, 1H). LCMS (ESI) 221 (M+H).

Example 42 Synthesis of5-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-2-amine, Compound 42

(2S,6R)-2,6-dimethyl-4-(6-nitro-3-pyridyl)morpholine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-2-amine in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, CHLOROFORM-d)δ ppm 1.20 (d, J=6.44 Hz, 6H) 2.27-2.39 (m, 2H) 3.11-3.21 (m, 2H)3.70-3.84 (m, 2H) 4.15 (s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.12 (dd, J=8.78,2.93 Hz, 1H) 7.72 (d, J=2.63 Hz, 1H). LCMS (ESI) 208 (M+H).

Example 43 Synthesis of5-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyridin-2-amine, Compound 43

(3S,5R)-3,5-dimethyl-1-(6-nitro-3-pyridyl)piperazine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyridin-2-amine in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, CHLOROFORM-d)δ ppm 1.09 (d, J=6.44 Hz, 6H) 2.20 (t, J=10.83 Hz, 2H) 2.95-3.08 (m, 2H)3.23 (dd, J=11.71, 2.05 Hz, 2H) 4.13 (s, 2H) 6.45 (d, J=8.78 Hz, 1H)7.14 (dd, J=8.78, 2.93 Hz, 1H) 7.73 (d, J=2.63 Hz, 1H). LCMS (ESI) 207(M+H).

Example 44 Synthesis of Compound 44

tert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate

A solution of intermediate A in ethanol (100 mL) was hydrogenated under30 psi of hydrogen using 10% Pd/C (0.7 g) in a pressure bomb for 7 hrs.After filtration of the reaction mixture through CELITE™, the organiclayer was concentrated under vacuum to afford tert-butylN-(2-amino-3-methyl-butyl) carbamate (3.8 g).

To a solution of 5-bromo-2,4-dichloro-pyrimidine (7.11 g, 0.0312 mole)in ethanol (100 mL) was added diisopropylethyl amine (5.45 mL, 1.0 eq)and tert-butyl N-(2-amino-3-methyl-butyl) carbamate (6.31 g, 0.0312mole). The reaction mixture was stirred at room temperature for 20 hrs.After concentration under vacuum, ethyl acetate and water were added.The organic layer was separated, dried with magnesium sulfate and thenconcentrated under vacuum. The crude product was purified by silica gelcolumn chromatography using hexane/ethyl acetate (0-30%) to affordtert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.77-0.85 (d, J=6.5 Hz, 3H)0.87 (d, J=6.73 Hz, 3H) 1.31-1.39 (m, 9H) 1.82-1.93 (m, 1H) 2.94 (d,J=5.56 Hz, 1H) 3.08-3.22 (m, 2H) 3.98 (d, J=8.20 Hz, 1H) 6.96 (d, J=8.78Hz, 1H) 8.21 (s, 1H). LCMS (ESI) 393 (M+H).

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-3-methyl-butyl]carbamate

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-3-methyl-butyl]carbamatewas synthesized by hosting tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto Sonogoshira conditions as described for tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamatefollowed by subsequent treatment with TBAF as described in the synthesisof tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.11 (d, J=6.44 Hz, 3H) 1.18 (t, J=7.03Hz, 6H) 1.21-1.26 (m, 12H) 2.88 (br. s., 1H) 3.43-3.78 (m, 6H) 3.97-4.08(m, 1H) 5.61 (s, 1H) 6.65 (s, 1H) 6.71-6.78 (m, 1H) 8.87 (s, 1H). LCMS(ESI) 441 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

To a solution tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamatein THE was added TBAF and the contents were heated at reflux for 3 hrs.Ethyl acetate and water were then added and the organic layer separated,dried with magnesium sulfate and then concentrated under vacuum. To thiscrude reaction was added acetic acid/water (9:1) and the contents werestirred for 12 hrs at room temperature. After concentration undervacuum, sat NaHCO₃ and ethyl acetate were added. The organic layer wasseparated, dried and then concentrated under vacuum. The crude reactionproduct thus obtained was dissolved in DMF, oxone was then added and thecontents stirred for 3 hrs. After addition of ethyl acetate, thereaction mixture was filtered through CELITE™ and concentrated undervacuum. Column chromatography of the crude product over silica gel usinghexane/ethyl acetate (0-100%) afforded7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=7.03 Hz, 3H) 0.97 (d,J=6.73 Hz, 3H) 1.52 (s, 9H) 1.99-2.23 (m, 1H) 3.98 (dd, J=14.05, 3.51Hz, 1H) 4.47-4.71 (m, 2H) 7.47 (s, 1H) 9.17 (s, 1H). LCMS (ESI) 383(M+H).

Compound 44

To7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.050 g, 0.00013 mole) in DCM (1.5 mL) was added DIC (32.7 mg) andDMAP (10 mg). The contents were stirred for 2 hrs. Trifluoroacetic acid(0.4 mL) was then added and stirring continued for an additional 30minutes. After addition of satd NaHCO₃ to neutralize the excess acid,ethyl acetate was added and the organic layer separated, dried usingmagnesium sulfate and then concentrated under vacuum. The crude productwas purified by silica gel column chromatography using hexane/ethylacetate (0-100%) to afford the product. ¹HNMR (600 MHz, DMSO-d₆) δ ppm0.72 (d, J=6.73 Hz, 3H) 0.97 (d, J=6.73 Hz, 3H) 2.09-2.22 (m, 1H) 3.57(dd, J=13.18, 4.98 Hz, 1H) 3.72 (dd, J=13.61, 4.25 Hz, 1H) 4.53 (dd,J=8.05, 3.95 Hz, 1H) 7.20 (s, 1H) 8.34 (d, J=4.98 Hz, 1H) 9.08 (s, 1H).LCMS (ESI) 265 (M+H).

Example 45 Synthesis of Compound 45

Compound 14 was hydrogenated with 10% Pd/C to afford the intermediatetert-butyl N-[(2R)-2-amino-3-methyl-butyl] carbamate, which was thentreated with 5-bromo-2,4-dichloro-pyrimidine using analogous reactionconditions as described for Compound 44 to afford Compound 45 Theanalytical data is consistent with that reported for the racemate(Intermediate 1A).

Example 46 Synthesis of Compound 46

Compound 15 was hydrogenated with 10% Pd/C to afford the intermediatetert-butyl N-[(2S)-2-amino-3-methyl-butyl]carbamate, which was thentreated with 5-bromo-2,4-dichloro-pyrimidine using analogous reactionconditions as described for Compound 44 to afford Compound 46. Theanalytical data (NMR and LCMS) was consistent with that reported for theracemate Compound 44.

Example 47 Synthesis of Compound 47

To a solution of Compound 44 (80 mg, 0.00030 mole) in DMF (3 mL) wasadded a 60% dispersion of sodium hydride in oil (40 mg). After stirringfor 15 minutes, methyl iodide (37 μL, 2 eq) was added. The contents werestirred at room temperature for 30 minutes. Saturated NaHCO₃ was thenadded followed by ethyl acetate. The organic layer was dried withmagnesium sulfate and then concentrated under vacuum to afford theproduct. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.74 (d, J=6.73 Hz, 3H) 0.91 (d,J=6.73 Hz, 3H) 2.04-2.20 (m, 1H) 3.04 (s, 3H) 3.69 (dd, J=13.76, 1.17Hz, 1H) 3.96 (dd, J=13.76, 4.68 Hz, 1H) 4.58 (dd, J=7.32, 3.51 Hz, 1H)7.16 (s, 1H) 9.05 (s, 1H). LCMS (ESI) 279 (M+H).

Example 48 Synthesis of Compound 48

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate

Compound 18 was hydrogenated with 10% Pd/C in ethanol under a blanket ofhydrogen at 50 psi in a pressure bomb to afford tert-butylN-[(2S)-2-amino-4-methyl-pentyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.91 (d, J=6.44 Hz, 3H) 0.94 (d,J=6.44 Hz, 3H) 1.32-1.51 (m, 11H) 1.55-1.67 (m, 1H) 3.28 (t, J=5.86 Hz,2H) 4.21-4.42 (m, 1H) 4.84 (s, 1H) 5.84 (d, J=7.32 Hz, 1H) 8.07 (s, 1H).LCMS (ESI) 407 (M+H).

To a solution of tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate(5.0 g, 12.3 mmole) in tolune (36 mL) and triethylamine (7.2 mL) wasadded under nitrogen, 3,3-diethoxyprop-1-yne (2.8 mL, 19.7 mmole),Pd₂(dba)₃ (1.1 g, 1.23 mmole), and triphenylarsine (3.8 g, 12.3 mmole).The contents were heated to 70 degrees for 24 hrs. After cooling to roomtemperature, the reaction mixture was filtered through CELITE™ and thenconcentrated under vacuum. The crude product was purified by silica gelcolumn chromatography using hexane/ethyl acetate (0-30%) to afford(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.88 (d, J=6.44 Hz, 3H) 0.97 (d,J=6.44 Hz, 3H) 1.47 (s, 9H) 1.49-1.54 (m, 1H) 1.56 (t, J=7.17 Hz, 2H)3.98 (dd, J=13.91, 3.07 Hz, 1H) 3.76 (dd, J=13.31, 4.13 Hz, 1H) 4.38 (d,J=14.05 Hz, 1H) 4.90 (t, J=7.17 Hz, 1H) 7.41 (s, 1H) 9.11 (s, 1H). LCMS(M+H) 397.

Compound 48 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d,J=6.73 Hz, 3H) 0.97 (d, J=6.44 Hz, 3H) 1.34-1.46 (m, 1H) 1.48-1.65 (m,2H) 3.40 (dd, J=13.32, 5.42 Hz, 1H) 3.76 (dd, J=13.47, 4.10 Hz, 1H)4.76-4.92 (m, 1H) 7.17 (s, 1H) 8.34 (d, J=5.27 Hz, 1H) 9.04 (s, 1H).LCMS (ESI) 279 (M+H).

Example 49 Synthesis of Compound 49

Compound 49 was synthesized in a manner similar to that described forCompound 47. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d, J=6.44 Hz, 3H) 0.97(d, J=6.44 Hz, 3H) 1.37-1.68 (m, 3H) 3.04 (s, 3H) 3.56 (d, J=13.47 Hz,1H) 4.00 (dd, J=13.32, 4.25 Hz, 1H) 4.82-4.94 (m, 1H) 7.16 (s, 1H) 9.03(s, 1H). LCMS (ESI) 293 (M+H).

Example 50 Synthesis of Compound 50

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-pentyl]carbamate

Compound 20 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-3-methyl-pentyl]carbamate which was reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-pentyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.88-0.95 (m, 6H) 1.11-1.20 (m, 1H)1.34 (s, 9H) 1.44-1.54 (m, 1H) 1.64-1.72 (m, 1H) 3.17-3.27 (m, 1H)3.33-3.43 (m, 1H) 4.11-4.21 (m, 1H) 4.81 (s, 1H) 5.92 (d, J=8.20 Hz, 1H)8.05 (s, 1H). LCMS (ESI) 407.

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3-methyl-pentyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3-methyl-pentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.76-0.89 (m, 6H) 1.03 (q, J=7.22 Hz, 3H)1.10-1.17 (m, 3H) 1.25-1.42 (m, 11H) 1.59-1.73 (m, 1H) 3.35-3.47 (m, 4H)3.51-3.73 (m, 2H) 3.99-4.11 (m, 1H) 5.52-5.56 (m, 1H) 6.76-7.03 (m, 2H)8.12-8.23 (m, 1H). LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.80 (t, J=7.47 Hz, 3H) 0.86 (d,J=7.03 Hz, 3H) 1.06-1.30 (m, 2H) 1.48 (s, 9H) 1.79-1.96 (m, 1H) 3.95(dd, J=14.05, 3.22 Hz, 1H) 4.52 (d, J=14.35 Hz, 1H) 4.61-4.73 (m, 1H)7.43 (s, 1H) 9.13 (s, 1H). LCMS (ESI) 397 (M+H).

Compound 50 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.74 (t,J=7.32 Hz, 3H) 0.89 (d, J=6.73 Hz, 3H) 1.00-1.12 (m, 2H) 1.82-1.94 (m,1H) 3.55 (dd, J=13.91, 4.83 Hz, 1H) 3.70 (dd, J=13.61, 4.25 Hz, 1H) 4.57(dd, J=7.91, 4.10 Hz, 1H) 7.17 (s, 1H) 8.31 (d, J=5.27 Hz, 1H) 9.05 (s,1H). LCMS (ESI) 279 (M+H).

Example 51 Synthesis of Compound 51

Compound 51 was synthesized in a manner similar to Compound 47. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.77 (t, J=7.47 Hz, 3H) 0.84 (d, J=6.73 Hz, 3H)1.07-1.16 (m, 2H) 1.82-1.95 (m, 1H) 3.03 (s, 3H) 3.68 (d, J=13.76 Hz,1H) 3.96 (dd, J=13.76, 4.39 Hz, 1H) 4.59-4.70 (m, 1H) 7.16 (s, 1H) 9.04(s, 1H). LCMS (ESI) 293 (M+H).

Example 52 Synthesis of Compound 52

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3,3-dimethyl-butyl]carbamate

Compound 21 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-3,3-dimethyl-butyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed using analogous reaction conditions as described fortert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3,3-dimethyl-butyl]carbamate.LCMS (ESI) 407 (M+H).

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3,3-dimethyl-butyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3,3-dimethyl-butyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2,2-dimethyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2,2-dimethyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 397 (M+H).

Intermediate 1F was synthesized using an analogous synthetic sequence asthat described for intermediate 1A. LCMS (ESI) 279 (M+H).

Example 53 Synthesis of Compound 53

Compound 53 was synthesized in a manner similar to that described forIntermediate 1CA. LCMS (ESI) 293 (M+H).

Example 54 Synthesis of Compound 54

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-phenyl-ethyl]carbamate

Compound 21 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-2-phenyl-ethyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-phenyl-ethyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.32 (s, 9H) 3.29-3.50 (m, 2H) 5.12-5.24(m, 1H) 7.10 (t, J=5.27 Hz, 1H) 7.21 (t, J=6.88 Hz, 1H) 7.26-7.34 (m,4H) 7.89 (d, J=7.32 Hz, 1H) 8.24 (s, 1H). LCMS (ESI) 427 (M+H).

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-phenyl-ethyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-phenyl-ethyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.14 (t, J=7.03 Hz, 6H) 1.32 (s, 9H) 3.39(s, 2H) 3.52-3.61 (m, 2H) 3.64-3.73 (m, 2H) 5.17-5.26 (m, 1H) 5.57 (s,1H) 7.07-7.14 (m, 1H) 7.20-7.25 (m, 1H) 7.26-7.33 (m, 4H) 7.90 (d,J=7.61 Hz, 1H) 8.19 (s, 1H). LCMS (ESI) 475 (M+H).

7-[(1S)-2-(tert-butoxycarbonylamino)-1-phenyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-2-(tert-butoxycarbonylamino)-1-phenyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 417 (M+H).

Compound 54

Compound 54 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 3.58-3.69(m, 1H) 4.13 (dd, J=13.47, 4.39 Hz, 1H) 6.07 (d, J=3.81 Hz, 1H) 6.85 (d,J=7.32 Hz, 2H) 7.19-7.31 (m, 3H) 7.34 (s, 1H) 8.27 (d, J=5.27 Hz, 1H)9.13 (s, 1H). LCMS (ESI) 299 (M+H).

Example 55 Synthesis of Compound 55

tert-butylN-[(1S)-1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]-2-methyl-propyl]

tert-butylN-[(1S)-1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]-2-methyl-propyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and Intermediate Eusing analogous reaction conditions as described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.95-1.02 (m, 6H) 1.35-1.45 (m, 9H)1.75-1.90 (m, 1H) 3.35-3.48 (m, 1H) 3.52-3.61 (m, 1H) 3.64-3.76 (m, 1H)4.56 (d, J=8.49 Hz, 1H) 6.47 (s, 1H) 8.07 (s, 1H). LCMS (ESI) 393 (M+H).

tert-butylN-[(1S)-1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]-2-methyl-propyl]carbamate

tert-butylN-[(1S)-1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]-2-methyl-propyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, CHLOROFORM-d) 6 ppm 0.90-1.00 (m, 6H) 1.18-1.25 (m, 6H)1.34-1.36 (m, 9H) 1.69-1.90 (m, 1H) 3.34-3.82 (m, 6H) 4.53-4.77 (m, 1H)5.45-5.55 (m, 1H) 6.37 (dd, J=15.37, 6.59 Hz, 1H) 6.56 (s, 1H) 8.05 (s,1H). LCMS (ESI) 441 (M+H).

7-[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.90 (d, J=6.73 Hz, 3H) 0.96(d, J=7.03 Hz, 3H) 1.55-1.66 (m, 10H) 4.14 (dd, J=13.61, 3.95 Hz, 1H)4.52-4.63 (m, 1H) 4.84 (dd, J=13.61, 1.32 Hz, 1H) 7.37 (s, 1H) 8.95 (s,1H). LCMS (ESI) 383 (M+H).

Compound 55

Compound 55 was synthesized using an analogous synthetic sequence asthat described for Compound 44. LCMS (ESI) 265 (M+H).

Example 56 Synthesis of Compound 56

Compound 56 was synthesized using 5-bromo-2,4-dichloro-pyrimidine andCompound 17 as starting materials, and following a similar sequence ofsynthetic steps as for Compound 55. The analytical data was consistentwith that described for its antipode (Compound 55). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.88 (d, J=6.44 Hz, 6H) 1.73-1.86 (m, 1H) 3.67-3.76 (m,2H) 4.11-4.21 (m, 1H) 7.13-7.19 (m, 1H) 8.56 (s, 1H) 9.05 (s, 1H). LCMS(ESI) 265 (M+H).

Example 57 Synthesis of Compound 57

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamate

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and tert-butylN-(2-amino-2-methyl-propyl)carbamate using analogous reaction conditionsas described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.LCMS (ESI) 379 (M+H).

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.11-1.22 (m, 6H) 1.31-1.45 (m, 15H)3.10-3.24 (m, 2H) 3.51-3.76 (m, 4H) 5.60 (s, 1H) 6.94 (s, 1H) 7.33 (t,J=6.44 Hz, 1H) 8.18 (s, 1H). LCMS (ESI) 427 (M+H).

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.43 (s, 9H) 1.73 (s, 6H) 4.06 (s,2H) 7.46 (s, 1H) 9.23 (s, 1H). LCMS (ESI) 369 (M+H).

Compound 57

Compound 57 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.73 (s,6H) 3.50 (d, J=2.93 Hz, 2H) 7.25 (s, 1H) 8.46-8.55 (m, 1H) 9.07 (s, 1H).LCMS (ESI) 251 (M+H).

Example 58 Synthesis of Compound 58

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclohexyl]methyl]carbamate

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclohexyl]methyl]carbamate was synthesized using 5-bromo-2,4-dichloro-pyrimidine andIntermediate K using the analogous reaction conditions as described fortert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.18-1.54 (m, 17H) 2.23 (d,J=14.35 Hz, 2H) 3.36 (d, J=6.44 Hz, 2H) 5.82 (s, 1H) 6.93 (s, 1H) 8.22(s, 1H). LCMS (ESI) 419 (M+H).

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclohexyl]methyl]carbamate

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclohexyl]methyl]carbamate was synthesized using similar experimental conditions to thoseused in the synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.08-1.16 (m, 6H) 1.17-1.54 (m, 17H) 2.13(br. s., 2H) 3.36 (d, J=6.73 Hz, 2H) 3.50-3.69 (m, 4H) 5.72 (s, 1H) 6.94(s, 1H) 5.72 (br. s., 1H) 8.17 (s, 1H). LCMS (ESI) 467 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]cyclohexyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]cyclohexyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.37-1.54 (m, 13H) 1.75 (br. s.,4H) 2.74 (br. s., 2H) 3.78-3.84 (m, 2H) 7.44-7.51 (m, 1H) 8.23 (s, 1H)9.11 (s, 1H). LCMS (ESI) 409 (M+H).

Compound 58

Compound 58 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.28 (br.s., 2H) 1.42 (br. s., 2H) 1.70 (br. s., 4H) 1.85-1.95 (m, 2H) 2.69 (m,2H) 7.16-7.25 (m, 1H) 8.41 (br. s., 1H) 9.04 (s, 1H). LCMS 291 (M+H).

Example 59 Synthesis of Compound 59

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate

tert-butyl N-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate was synthesized using 5-bromo-2,4-dichloro-pyrimidineand Intermediate L using analogous reaction conditions as described fortert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.34 (s, 9H) 1.50-1.58 (m, 2H) 1.63-1.78(m, 4H) 1.96-2.06 (m, 2H) 3.25 (d, J=6.15 Hz, 2H) 6.71 (s, 1H) 7.18 (t,J=6.29 Hz, 1H) 8.20 (s, 1H). LCMS (ESI) 405 (M+H).

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamate

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 453 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.47 (s, 9H) 1.74 (br. s., 2H) 1.88(br. s., 2H) 2.04 (br. s., 2H) 2.41-2.45 (m, 2H) 4.06 (s, 2H) 7.45 (s,1H) 9.11 (s, 1H). LCMS (ESI) 395 (M+H).

Compound 59

Compound 59 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.72 (br.s., 2H) 1.86-1.93 (m, 2H) 1.99 (d, J=3.81 Hz, 2H) 2.40 (br. s., 2H) 3.48(d, J=2.34 Hz, 2H) 7.22 (s, 1H) 8.53 (br. s., 1H) 9.05 (s, 1H). LCMS(ESI) 277 (M+H).

Example 60 Synthesis of Compound 60

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl] carbamate

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and Intermediate Busing analogous reaction conditions as described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.The analytical data is consistent with that described for theL-enantiomer.

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-4-methyl-pentyl]carbamate

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-4-methyl-pentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 1.21-1.31 (m, 12H) 1.38-1.46 (m,11H) 1.70 (m, 1H) 3.24 (m, 2H) 3.65-3.82 (m, 4H) 4.86 (br s., 1H), 5.65(s, 1H) 5.85 (br s., 1H) 6.94 (s, 1H) 8.21 (s, 1H). LCMS (ESI) 455(M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. The analytical data was consistent with that described for theL-isomer.

Compound 60

Compound 60 was synthesized using an analogous synthetic sequence asthat described for Compound 44. The analytical data was consistent withthat described for the L-isomer.

Example 61 Synthesis of Compound 61

To a solution of Compound 60 (100 mg, 0.00024 mole) in DMF (3.0 mL) wasadded sodium hydride (60% dispersion in oil), (27.6 mg, 3 eq). Afterstirring for 15 mins, methyl iodide (30, 2 eq) was added. The contentswere stirred at room temperature for 30 mins. After the addition of satNaHCO₃, ethyl acetate was added. Separation of the organic layerfollowed by drying with magnesium sulfate and concentration under vacuumafforded the product. Analytical data was similar to the Compound 49.

Example 62 Synthesis of Compound 62

tert-butylN-[(1S,2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]carbamate

tert-butylN-[(1S,2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[(1S,2S)-2-aminocyclopentyl]carbamate with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.27 (s, 9H) 1.42-1.54 (m, 2H) 1.56-1.65(m, 2H) 1.80-1.88 (m, 1H) 1.96-2.01 (m, 1H) 3.88-3.96 (m, 1H) 4.03-4.09(m, 1H) 6.91 (d, J=8.20 Hz, 1H) 7.41 (d, J=7.32 Hz, 1H) 8.18 (s, 1H).LCMS (ESI) 391 (M+H).

tert-butylN-[(1S,2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]carbamate

tert-butylN-[(1S,2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.13 (t, 6H) 1.28 (s, 9H) 1.42-1.52 (m,2H) 1.58-1.65 (m, 2H) 1.81-1.90 (m, 1H) 1.99-2.08 (m, 1H) 3.49-3.60 (m,2H) 3.63-3.71 (m, 2H) 3.84-3.93 (m, 1H) 3.96-4.04 (m, 1H) 5.53 (s, 1H)6.96 (d, J=7.90 Hz, 1H) 7.34 (d, J=7.03 Hz, 1H) 8.14 (s, 1H). LCMS (ESI)439 (M+H).

7-[(1S,2S)-2-(tert-butoxycarbonylamino)cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S,2S)-2-(tert-butoxycarbonylamino)cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.41-1.52 (m, 9H) 1.55-1.68 (m, 1H)1.88-2.00 (m, 2H) 2.05-2.15 (m, 1H) 2.26-2.35 (m, 1H) 2.71-2.89 (m, 1H)4.01-4.16 (m, 1H) 4.28-4.45 (m, 1H) 7.41 (s, 1H) 9.11 (s, 1H). LCMS(ESI) 381 (M+H).

Compound 62

Compound 62 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.48-1.60(m, 1H) 1.88-1.98 (m, 3H) 1.99-2.08 (m, 1H) 2.66-2.75 (m, 1H) 3.63-3.74(m, 1H) 3.99-4.12 (m, 1H) 7.21 (s, 1H) 8.89 (s, 1H) 9.04 (s, 1H). LCMS(ESI) 263 (M+H).

Example 63 Synthesis of Compound 63

To chloro tricycliclactam (0.050 g, 0.225 mmole) in dioxane (2.0 mL)under nitrogen was added 5-(4-methylpiperazin-1-yl)pyridin-2-amine(0.052 g, 1.2 eq, 0.270 mmole) followed by the addition of Pd₂(dba)₃(18.5 mg), BINAP (25 mg) and sodium-tert-butoxide (31 mg, 0.324 mmole).The contents of the flask are degassed for 10 minutes and then heated to100 degrees for 12 hours. The crude reaction was loaded on a silica gelcolumn and eluted with DCM/MeOH (0-15%) to afford the desired product(26 mg). To this compound dissolved in DCM/MeOH (10%) was added 3N HClin iso-propanol (2 eq) and the reaction was stirred overnight.Concentration under vacuum afforded the hydrochloride salt. ¹HNMR(d6-DMSO) δ ppm 11.13 (brs, 1H), 9.07 (s, 1H), 8.42 (s, 1H), 8.03 (br m1H), 7.99 (s, 1H), 7.67 (brm, 1H), 7.18 (s, 1H), 4.33 (m, 2H), 3.79 (m,2H), 3.64 (m, 2H), 3.50 (m, 2H), 3.16 (m, 4H), 2.79 (s, 3H). LCMS (ESI)379 (M+H).

Example 64 Synthesis of Compound 64

To chloro tricycliclactam (0.075 g, 0.338 mmole) in dioxane (3.5 mL)under nitrogen was added tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate (0.098 g, 1.05 eq)followed by the addition of Pd₂(dba)₃ (27 mg), BINAP (36 mg) andsodium-tert-butoxide (45 mg). The contents were heated at reflux for 11hrs. The crude reaction was loaded onto a silica gel column and elutedwith DCM/MeOH (0-10%) to afford the desired product (32 mg). ¹HNMR(d6-DMSO) δ ppm 9.48 (s, 1H), 8.84 (s, 1H), 8.29 (s, 1H), 8.18 (s, 1H),7.99 (s, 1H), 7.42 (m, 1H), 6.98 (s, 1H), 4.23 (m, 2H), 3.59 (m, 2H),3.45 (m, 4H), 3.50 (m, 2H), 3.05 (m, 4H). LCMS (ESI) 465 (M+H).

Example 65 Synthesis of Compound 65

To a solution of Compound 64 (23 mg) in 10% DCM/MeOH was added 10 mL ofa 3M solution of HCl in iso-propanol. The contents were stirred for 16hrs. Concentration of the reaction mixture afforded the hydrochloridesalt. ¹HNMR (d6-DMSO) δ ppm 9.01 (s, 1H), 7.94 (m, 1H), 7.86 (m, 1H),7.23 (s, 1H), 4.30 (m, 2H), 3.64 (m, 2H), 3.36 (m, 4H), 3.25 (m, 4H).LCMS (ESI) 465 (M+H).

Example 66 Synthesis of Compound 66

To chloro-N-methyltricyclic amide (0.080 g, 0.338 mmole) in dioxane (3.5mL) under nitrogen was added tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate 0.102 g (1.1 eq) followedby the addition of Pd₂(dba)₃ (27 mg), BINAP (36 mg) andsodium-tert-butoxide (45 mg). The contents were heated at reflux for 11hrs. The crude product was purified using silica gel columnchromatography with an eluent of dichloromethane/methanol (0-5%) toafford the desired product (44 mg). ¹HNMR (d6-DMSO) δ ppm 9.49 (s, 1H),8.85 (s, 1H), 8.32 (m, 1H), 8.02 (s, 1H), 7.44 (m, 1H), 7.00 (s, 1H),4.33 (m, 2H), 3.80 (m, 2H), 3.48 (m, 4H), 3.07 (m, 4H), 3.05 (s, 3H),1.42 (s, 9H). LCMS (ESI) 479 (M+H).

Example 67 Synthesis of Compound 67

To Compound 66 (32 mg) was added 3N HCL (10 mL) in isopropanol and thecontents were stirred at room temperature overnight for 16 hrs.Concentration afforded the hydrochloride salt. ¹HNMR (d6-DMSO) δ ppm9.13 (m, 2H), 8.11 (m, 1H), 8.10 (s, 1H), 7.62 (m, 1H), 7.21 (s, 1H),4.43 (m, 2H), 3.85 (m, 2H), 3.41 (m, 4H), 3.28 (m, 4H), 3.08 (s, 3H).LCMS (ESI) 379 (M+H).

Example 68 Synthesis of Compound 68

Compound 68 was synthesized using similar experimental conditions tothat described for compound 64. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.79 (d,J=7.03 Hz, 3H) 1.01 (d, J=6.73 Hz, 3H) 1.35-1.48 (m, 9H) 2.16 (dd,J=14.64, 6.73 Hz, 1H) 3.00-3.14 (m, 4H) 3.40-3.51 (m, 4H) 3.51-3.60 (m,1H) 3.63-3.74 (m, 1H) 4.44 (dd, J=7.90, 3.81 Hz, 1H) 6.99 (s, 1H) 7.46(dd, J=8.93, 2.78 Hz, 1H) 7.94-8.09 (m, 2H) 8.31 (dd, J=9.08, 1.46 Hz,1H) 8.85 (s, 1H) 9.46 (s, 1H). LCMS (ESI) 507 (M+H).

Example 69 Synthesis of Compound 69

Compound 69 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.77-0.86 (m, 3H) 0.96 (d, J=7.03 Hz, 3H)2.10-2.24 (m, 1H) 3.07 (s, 3H) 3.37-3.79 (m, 8H) 4.00 (dd, J=13.61, 4.54Hz, 2H) 4.63-4.73 (m, 1H) 7.20 (s, 1H) 7.58-7.71 (m, 1H) 7.99 (d, J=2.34Hz, 1H) 8.12 (d, J=9.37 Hz, 1H) 9.11 (s, 1H) 9.41 (br. s., 2H) 11.76(br. s., 1H). LCMS (ESI) 421 (M+H).

Example 70 Synthesis of Compound 70

Compound 70 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. The characterization data (NMR and LCMS) was consistent with thatreported for compound 71.

Example 71 Synthesis of Compound 71

Compound 71 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.79 (d, J=6.73 Hz, 3H) 1.01 (d,J=6.73 Hz, 3H) 2.18 (dd, J=14.49, 7.17 Hz, 1H) 3.18-3.84 (m, 10H)4.53-4.71 (m, 1H) 7.24 (s, 1H) 7.65 (d, J=9.37 Hz, 1H) 8.01 (d, J=2.64Hz, 1H) 8.14 (d, J=1.46 Hz, 1H) 8.35 (d, J=5.27 Hz, 1H) 9.14 (s, 1H)9.46 (s, 2H) 11.80 (s, 1H) LCMS (ESI) 407 (M+H).

Example 72 Synthesis of Compound 72 (Compound UUU)

Compound 72 was synthesized using similar experimental conditions tothat described for compounds 64 and 65 and was recovered as an HCl salt.¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.77 (d, J=7.03 Hz, 3H) 0.99 (d, J=6.73Hz, 3H) 2.10-2.24 (m, 1H) 3.18-3.81 (m, 10H) 4.54-4.69 (m, 1H) 7.22 (s,1H) 7.63 (d, J=9.08 Hz, 1H) 7.99 (d, J=2.63 Hz, 1H) 8.11 (s, 1H) 8.33(d, J=5.27 Hz, 1H) 9.12 (s, 1H) 9.43 (s, 2H) 11.77 (s, 1H). LCMS (ESI)407 (M+H).

Example 73 Synthesis of Compound 73

Compound 73 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.84 (d, J=6.73 Hz, 3H) 0.98 (d,J=6.73 Hz, 3H) 2.12-2.26 (m, 1H) 3.09 (s, 3H) 3.22-3.81 (m, 8H) 4.01(dd, J=13.61, 4.25 Hz, 2H) 4.59-4.72 (m, 1H) 7.19 (s, 1H) 7.74 (s, 1H)7.96-8.10 (m, 2H) 9.08 (s, 1H) 9.22 (s, 2H). LCMS (ESI) 421 (M+H).

Example 74 Synthesis of Compound 74

Compound 74 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=4.98 Hz, 3H) 0.95 (d, J=4.98 Hz, 3H)1.42-1.70 (m, 3H) 2.77 (d, J=2.93 Hz, 3H) 3.07-4.14 (m, 10H) 4.95 (s,1H) 7.20 (s, 1H) 7.66 (d, J=9.66 Hz, 1H) 7.94 (s, 1H) 8.08-8.16 (m, 1H)8.33 (d, J=4.68 Hz, 1H) 9.09 (s, 1H) 11.38 (s, 1H) 11.71 (s, 1H). LCMS(ESI) 435 (M+H).

Example 75 Synthesis of Compound 75

Compound 75 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.87 (d, J=6.15 Hz, 3H) 0.94 (d,J=6.15 Hz, 3H) 1.57 (d, J=84.61 Hz, 3H) 3.05 (s, 3H) 3.13-3.55 (m, 8H)3.69 (d, J=78.17 Hz, 2H) 4.90 (s, 1H) 7.15 (s, 1H) 7.63-7.85 (m, 1H)7.93 (s, 1H) 8.26 (s, 1H) 9.03 (s, 1H) 9.20 (s, 2H). LCMS (ESI) 421(M+H).

Example 76 Synthesis of Compound 76

Compound 76 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=6.44 Hz, 3H) 0.95 (d, J=6.44 Hz, 3H)1.43-1.70 (m, 3H) 2.78 (d, J=2.93 Hz, 3H) 3.05 (s, 3H) 3.24-3.84 (m, 8H)4.01 (d, J=9.66 Hz, 2H) 4.89-5.01 (m, 1H) 7.15 (s, 1H) 7.77 (s, 1H)7.91-8.05 (m, 2H) 9.03 (s, 1H) 10.96-11.55 (m, 2H). LCMS (ESI) 449(M+H).

Example 77 Synthesis of Compound 77

Compound 77 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.83-0.88 (d, J=6.15 Hz, 3H) 0.95(d, J=6.15 Hz, 3H) 1.40-1.71 (m, 3H) 3.28-3.83 (m, 8H) 4.00 (d, J=3.22Hz, 2H) 4.91-5.08 (m, 1H) 7.17 (s, 1H) 7.68 (d, J=9.66 Hz, 1H) 7.93 (s,1H) 8.07 (s, 1H) 9.06 (s, 1H) 9.40 (s, 2H) 11.59 (s, 1H). LCMS (ESI) 435(M+H).

Example 78 Synthesis of Compound 78

To Compound 50 0.060 g (0.205 mmole) was added5-(4-methylpiperazin-1-yl)pyridin-2-amine (35.42 mg, 0.9 eq) followed bythe addition of 1,4-dioxane (3 mL). After degassing with nitrogen,Pd₂dba₃ (12 mg), BINAP (16 mg) and sodium tert-butoxide (24 mg) wereadded. The contents were then heated at 90 degrees in a CEM Discoverymicrowave for 3 hrs. The reaction was then loaded onto a silica gelcolumn and purified by eluting with DCM/MeOH (0-15%). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.75 (t, J=7.47 Hz, 3H) 0.91 (d, J=6.73 Hz, 3H) 1.04-1.20(m, 2H) 1.80-1.98 (m, 1H) 2.77 (d, J=3.81 Hz, 3H) 2.94-3.90 (m, 10H)4.54-4.68 (m, 1H) 7.06-7.23 (m, 2H) 7.56-7.75 (m, 1H) 7.90-8.12 (m, 2H)8.29 (s, 1H) 9.07 (s, 1H) 10.98-11.74 (m, 2H). LCMS (ESI) 435 (M+H).

Example 79 Synthesis of Compound 79

Compound 79 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.75(t, J=7.32 Hz, 3H) 0.90 (d, J=6.73 Hz, 3H) 1.07-1.15 (m, 2H) 1.85-1.94(m, 1H) 3.17-3.75 (m, 10H) 4.58-4.67 (m, 1H) 7.17 (s, 1H) 7.71 (s, 1H)7.96 (s, 1H) 7.98-8.05 (m, 1H) 8.28 (d, J=4.10 Hz, 1H) 9.06 (s, 1H) 9.39(s, 2H). LCMS (ESI) 421 (M+H).

Example 80 Synthesis of Compound 80

Compound 80 was synthesized in a similar manner to that described forcompound 78. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.78 (t, J=7.32 Hz, 3H) 0.86(d, J=6.73 Hz, 3H) 1.13-1.21 (m, 2H) 1.84-1.96 (m, 1H) 2.77 (d, J=4.39Hz, 3H) 3.04 (s, 3H) 3.11-3.84 (m, 8H) 3.98 (dd, J=13.61, 4.25 Hz, 2H)4.66-4.74 (m, 1H) 7.17 (s, 1H) 7.64 (s, 1H) 7.96 (d, J=2.34 Hz, 1H)8.03-8.13 (m, 1H) 9.08 (s, 1H) 11.26 (s, 1H) 11.66 (s, 1H). LCMS (ESI)449 (M+H).

Example 81 Synthesis of Compound 81

The compound was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.78(t, J=7.32 Hz, 3H) 0.85 (d, J=6.73 Hz, 3H) 1.10-1.27 (m, 2H) 1.82-1.99(m, 1H) 3.04 (s, 3H) 3.28-3.77 (m, 8H) 3.97 (dd, J=13.91, 4.54 Hz, 2H)4.62-4.75 (m, 1H) 7.07-7.24 (m, 1H) 7.62-7.75 (m, 1H) 7.94 (d, J=2.34Hz, 1H) 7.97-8.08 (m, 1H) 9.05 (s, 1H) 9.29 (s, 2H). LCMS (ESI) 435(M+H).

Example 82 Synthesis of Compound 82

The compound was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.96(s, 9H) 3.15-3.87 (m, 10H) 4.42-4.53 (m, 1H) 6.99 (s, 1H) 7.24 (s, 1H)8.06 (s, 1H) 8.11-8.21 (m, 1H) 8.79-8.98 (m, 2H) 9.25 (s, 2H) 9.88 (s,1H). LCMS (ESI) 421 (M+H).

Example 83 Synthesis of Compound 83

Compound 83 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.95(s, 9H) 2.79 (d, J=4.10 Hz, 3H) 3.06-3.86 (m, 10H) 4.56-4.67 (m, 1H)7.17 (s, 1H) 7.70 (s, 1H) 7.96 (d, J=2.63 Hz, 1H) 7.99-8.08 (m, 1H) 8.26(s, 1H) 9.06 (s, 1H) 10.80 (s, 1H). LCMS (ESI) 435 (M+H).

Example 84 Synthesis of Compound 84

Compound 84 was synthesized in a similar manner to that described forcompound 78 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δppm 2.75-2.81 (m, 3H) 3.12-3.16 (m, 2H) 3.46-3.54 (m, 4H) 3.60-3.69 (m,2H) 3.72-3.79 (m, 1H) 4.07-4.18 (m, 2H) 6.06-6.09 (m, 1H) 6.90 (d,J=7.61 Hz, 2H) 7.20-7.31 (m, 3H) 7.33 (s, 1H) 7.49-7.55 (m, 1H)7.62-7.70 (m, 1H) 7.92 (d, J=2.93 Hz, 1H) 8.22 (s, 1H) 9.14 (s, 1H).LCMS (ESI) 455 (M+H).

Example 85 Synthesis of Compound 85

Compound 85 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 3.21(s, 4H) 3.35-3.67 (m, 5H) 4.07-4.20 (m, 2H) 6.13 (s, 1H) 6.90 (d, J=7.32Hz, 2H) 7.22-7.31 (m, 3H) 7.36 (s, 1H) 7.48 (d, J=9.37 Hz, 1H) 7.93 (d,J=2.34 Hz, 1H) 8.04-8.11 (m, 1H) 8.25 (d, J=4.98 Hz, 1H) 9.17 (s, 1H)11.77 (br, s., 1H). LCMS (ESI) 441 (M+H).

Example 86 Synthesis of Compound 86

Compound 86 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.90(d, J=6.15 Hz, 6H) 1.72-1.89 (m, 1H) 3.15-3.92 (m, 9H) 4.10-4.46 (m, 2H)7.18 (s, 1H) 7.59 (d, J=8.78 Hz, 1H) 8.00 (s, 1H) 8.13 (d, J=9.37 Hz,1H) 8.55 (s, 1H) 9.09 (s, 1H) 9.67 (s, 2H) 11.91 (s, 1H). LCMS (ESI) 407(ESI).

Example 87 Synthesis of Compound 87

Compound 87 was synthesized in a manner similar to compound 86 and wasconverted to an HCl salt. The characterization data (NMR and LCMS) wassimilar to that obtained for the antipode compound 86.

Example 88 Synthesis of Compound 88

Compound 88 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.78(s, 6H) 3.40-3.53 (m, 6H) 3.64-3.73 (m, 4H) 7.27 (s, 1H) 7.66 (d, J=9.37Hz, 1H) 7.98 (d, J=2.34 Hz, 1H) 8.12 (br. s., 1H) 8.47 (br. s., 1H) 9.11(s, 1H) 9.45 (br. s., 2H) 11.62 (br. s., 1H). LCMS (ESI) 393 (M+H).

Example 89 Synthesis of Compound 89 (also referred to as Compound T)

Compound 89 was synthesized in a similar manner to that described forcompound 78 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.47 (br. s., 6H) 1.72 (br. s., 2H) 1.92 (br. s., 2H) 2.77 (br. s.,3H) 3.18 (br. s., 2H) 3.46 (br. s., 2H) 3.63 (br. s., 2H) 3.66 (d,J=6.15 Hz, 2H) 3.80 (br. s., 2H) 7.25 (s, 1H) 7.63 (br. s., 2H) 7.94(br. s., 1H) 8.10 (br. s., 1H) 8.39 (br. s., 1H) 9.08 (br. s., 1H) 11.59(br. s., 1H). LCMS (ESI) 447 (M+H).

Example 90 Synthesis of Compound 90 (also referred to as Compound Q)

Compound 90 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.27-1.64 (m, 6H) 1.71 (br. s., 2H) 1.91 (br. s., 2H) 2.80 (br. s., 1H)3.17-3.24 (m, 2H) 3.41 (br. s., 4H) 3.65 (br. s., 4H) 7.26 (br. s., 1H)7.63 (br. s., 1H) 7.94 (br. s., 1H) 8.13 (br. s., 1H) 8.40 (br. s., 1H)9.09 (br. s., 1H) 9.62 (br. s., 1H) 11.71 (br. s., 1H). LCMS (ESI) 433(M+H).

Example 91 Synthesis of Compound 91 (also referred to as Compound ZZ)

Compound 91 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.64-1.75 (m, 2H) 1.83-1.92 (m, 2H) 1.96-2.06 (m, 2H)2.49-2.58 (m, 2H) 2.79 (d, J=3.81 Hz, 3H) 3.06-3.18 (m, 4H) 3.59-3.69(m, 2H) 3.73-3.83 (m, 2H) 4.04-4.12 (m, 2H) 7.17 (br. s., 1H) 7.60-7.70(m, 2H) 7.70-7.92 (m, 2H) 7.96 (br. s., 1H) 8.41 (br. s., 1H) 8.98 (br.s., 1H) 10.77 (br. s., 1H). LCMS (ESI) 433 (M+H).

Example 92 Synthesis of Compound 92

Compound 92 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.64-1.75 (m, 2H) 1.84-1.92 (m, 2H) 1.96-2.05 (m, 2H) 2.48-2.56 (m, 2H)3.22 (br. s., 4H) 3.42-3.48 (m, 4H) 3.60-3.69 (m, 2H) 4.05-4.13 (m, 1H)7.18 (s, 1H) 7.65 (d, J=13.47 Hz, 1H) 7.70-7.77 (m, 1H) 7.94 (d, J=1.76Hz, 1H) 8.42 (br. s., 1H) 9.00 (s, 1H) 9.15 (br. s., 2H). LCMS (ESI) 419(M+H).

Example 93 Synthesis of Compound 93

Compound 93 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.76(br. s., 2H) 1.89 (br. s., 2H) 2.03 (br. s., 2H) 2.47-2.58 (m, 2H) 3.04(s, 3H) 3.22 (br. s., 4H) 3.39 (br. s., 4H) 3.66 (s, 2H) 7.21 (s, 1H)7.67 (d, J=9.37 Hz, 1H) 7.93 (br. s., 1H) 7.98-8.09 (m, 1H) 9.04 (s, 1H)9.34 (br. s., 2H) 11.31 (br. s., 1H). LCMS (ESI) 433 (M+H).

Example 94 Synthesis of Compound 94

Compound 94 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.66-1.77 (m, 2H) 1.84-1.94 (m, 2H) 1.96-2.08 (m, 2H)2.48-2.57 (m, 2H) 3.36-3.52 (m, 4H) 3.60-3.80 (m, 6H) 7.21 (s, 1H)7.53-7.74 (m, 2H) 7.86 (s, 1H) 8.02 (s, 1H) 8.45 (s, 1H) 9.03 (s, 1H)11.19 (br. s., 1H). LCMS (ESI) 420 (M+H).

Example 95 Synthesis of Compound 95

Compound 95 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.65-1.79 (m, 2H) 1.85-1.95 (m, 2H) 1.97-2.08 (m, 2H)2.47-2.54 (m, 2H) 3.40-3.58 (m, 5H) 3.65 (dd, J=21.67, 5.56 Hz, 1H)3.69-3.78 (m, 4H) 7.24 (s, 1H) 7.97-8.17 (m, 2H) 8.48 (s, 1H) 9.08 (s,1H) 11.81 (s, 1H). LCMS (ESI) 421 (M+H).

Example 96 Synthesis of Compound 96

Compound 96 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.55-1.74 (m, 2H) 1.80-1.98 (m, 4H) 2.48-2.60 (m, 2H)3.40-3.50 (m, 4H) 3.57-3.72 (m, 2H) 3.90-4.20 (m, 4H) 7.08 (s, 1H)7.37-7.57 (m, 2H) 7.70 (m, 2H) 8.32 (s, 1H) 8.88 (s, 1H) 9.98 (s, 1H).LCMS (ESI) 419 (M+H).

Example 97 Synthesis of Compound 97 (also referred to as Compound III)

Compound 97 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.30 (d, J=5.27 Hz, 6H) 1.65-1.78 (m, 2H) 1.83-1.95 (m,2H) 1.97-2.10 (m, 2H) 2.45-2.55 (m, 2H) 3.25-3.36 (m, 1H) 3.39-3.48 (m,4H) 3.60-3.70 (m, 4H) 3.75-4.15 (m, 2H) 7.24 (s, 1H) 7.54-7.75 (m, 2H)7.95 (s, 1H) 8.10 (s, 1H) 8.49 (s, 1H) 9.07 (s, 1H) 11.25 (s, 1H) 11.48(s, 1H). LCMS (ESI) 461 (M+H).

Example 98 Synthesis of Compound 98

Compound 98 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.99 (d, J=6.15 Hz, 6H) 1.65-1.78 (m, 2H) 1.90 (m, 2H)1.97-2.08 (m, 2H) 2.08-2.17 (m, 1H) 2.45-2.55 (m, 2H) 2.88-3.02 (m, 2H)3.33-3.48 (m, 4H) 3.50-3.90 (m, 6H) 7.24 (s, 1H) 7.67 (s, 2H) 7.94 (s,1H) 8.12 (s, 1H) 8.49 (s, 1H) 9.07 (s, 1H) 10.77 (s, 1H) 11.51 (s, 1H).LCMS (ESI) 475 (M+H).

Example 99 Synthesis of Compound 99

Compound 99 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.13 (d, J=5.86 Hz, 6H) 1.66-1.77 (m, 2H) 1.84-1.94 (m,2H) 1.97-2.09 (m, 2H) 2.40-2.53 (m, 2H) 3.37-3.49 (m, 2H) 3.50-3.59 (m,2H) 3.59-3.73 (m, 4H) 7.23 (s, 1H) 7.64 (m, 3H) 7.85 (s, 1H) 8.11 (s,1H) 8.47 (s, 1H) 9.05 (s, 1H). 11.35 (br s., 1H). LCMS (ESI) 448 (M+H).

Example 100 Synthesis of Compound 100

Compound 100 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.50-1.57 (m, 2H) 1.62-1.68 (m, 3H) 1.68-1.75 (m, 2H)1.84-1.92 (m, 2H) 1.97-2.08 (m, 2H) 2.48-2.53 (m, 2H) 3.14-3.23 (m, 4H)3.43-3.47 (m, 2H) 3.58-3.70 (m, 2H) 7.22 (s, 1H) 7.58-7.70 (m, 2H)7.85-8.00 (m, 1H) 8.16 (d, 1H) 8.46 (s, 1H) 9.04 (s, 1H) 11.37 (br s.,1H). LCMS (ESI) 418 (M+H).

Example 101 Synthesis of Compound 101 (also referred to as Compound WW)

Compound 101 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.72 (s, 2H) 1.90 (s, 4H) 2.03 (s, 2H) 2.21 (s, 2H)2.48-2.54 (m, 2H) 2.73 (s, 2H) 3.03 (s, 2H) 3.25-3.35 (m, 1H) 3.38-3.48(m, 4H) 3.65-3.99 (m, 5H) 7.23 (s, 1H) 7.63 (d, J=9.66 Hz, 1H) 7.90 (s,1H) 8.13 (s, 1H) 8.47 (s, 1H) 9.06 (s, 1H) 10.50 (br s., 1H). LCMS (ESI)503 (M+H).

Example 102 Synthesis of Compound 102 (also referred to as Compound HHH)

Compound 102 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.63-1.85 (m, 6H) 1.87-1.92 (m, 2H) 1.99-2.06 (m, 2H)2.15-2.23 (m, 2H) 2.47-2.53 (m, 1H) 2.69-2.79 (m, 2H) 2.81-2.91 (m, 2H)2.98-3.08 (m, 2H) 3.32-3.48 (m, 4H) 3.57-3.72 (m, 4H) 3.77-3.85 (m, 2H)7.22 (s, 1H) 7.60-7.68 (m, 2H) 7.90 (s, 1H) 8.07 (s, 1H) 8.46 (s, 1H)9.04 (s, 1H). 11.41 (br s., 1H). LCMS (ESI) 501 (M+H).

Example 103 Synthesis of Compound 103

Compound 103 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.64-1.76 (m, 2H) 1.87-1.93 (m, 2H) 2.00-2.07 (m, 2H)2.48-2.53 (m, 2H) 2.67-2.72 (m, 4H) 3.44-3.47 (m, 2H) 3.50-3.55 (m, 4H)7.24 (s, 1H) 7.61 (d, J=9.37 Hz, 2H) 7.86 (d, J=2.63 Hz, 1H) 8.09 (d,J=12.88 Hz, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 11.41 (br s., 1H). LCMS (ESI)436 (M+H).

Example 104 Synthesis of Compound 104

Compound 104 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.29 (d, J=6.73 Hz, 6H) 1.66-1.79 (m, 2H) 1.84-1.95 (m,2H) 1.98-2.09 (m, 2H) 2.46-2.55 (m, 2H) 3.29-3.39 (m, 2H) 3.58-3.70 (m,4H) 3.77-3.86 (m, 4H) 7.24 (s, 1H) 7.66 (d, J=9.37 Hz, 1H) 7.96 (d,J=2.93 Hz, 1H) 8.08 (s, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 9.28 (s, 1H) 9.67(s, 1H) 11.36 (s, 1H). LCMS (ESI) 447 (M+H).

Example 105 Synthesis of Compound 105

Compound 105 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.73 (s, 2H) 1.76-1.85 (m, 2H) 1.85-1.94 (m, 2H)1.98-2.07 (m, 2H) 2.19-2.26 (m, 2H) 2.48-2.52 (m, 1H) 2.70-2.81 (m, 4H)3.13-3.20 (m, 1H) 3.30-3.48 (m, 3H) 3.58-3.71 (m, 4H) 3.78-3.84 (m, 4H)7.24 (s, 1H) 7.62 (d, J=9.37 Hz, 2H) 7.89 (d, J=1.17 Hz, 1H) 8.09-8.18(m, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 11.46 (br s., 1H). LCMS (ESI) 519(M+H).

Example 106 Synthesis of Compound 106

Compound 106 was synthesized using similar conditions to those describedfor compound 78 followed by the deblocking step described for compound65 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.65-1.75 (m, 2H) 1.85-1.93 (m, 2H) 1.93-1.99 (m, 1H) 2.00-2.06 (m, 2H)2.08-2.14 (m, 1H) 2.47-2.55 (m, 2H) 3.07-3.25 (m, 2H) 3.25-3.69 (m, 5H)4.46 (s, 1H) 4.67 (s, 1H) 7.22 (s, 1H) 7.58-7.69 (m, 2H) 8.46 (s, 1H)9.02 (s, 1H) 9.34 (s, 1H) 9.65 (s, 1H). LCMS (ESI) 431 (M+H).

Example 107 Synthesis of Compound 107 (also referred to as Compound YY)

Compound 107 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.65-1.82 (m, 3H) 1.89 (br. s., 2H) 1.98-2.08 (m, 2H)2.13 (br. s., 2H) 2.47-2.55 (m, 2H) 2.68 (d, J=4.98 Hz, 6H) 2.71-2.80(m, 2H) 3.29-3.71 (m, 10H) 7.16-7.26 (m, 1H) 7.67 (d, J=9.66 Hz, 2H)7.91 (d, J=2.05 Hz, 1H) 8.14 (br. s., 1H) 8.48 (br. s., 1H) 9.05 (s, 1H)11.14 (br. s., 1H) 11.43 (br. s., 1H). LCMS (ESI) 461 (M+H).

Example 108 Synthesis of Compound 108

Compound 108 was synthesized in a manner similar to that described forcompounds 64 and 65 and was recovered as an HCl salt. The analyticaldata was consistent with that described for the antipode compound 75.

Example 109 Synthesis of Compound 109

Compound 109 was synthesized in a manner similar to that described forcompounds 64 and 65 and was recovered as an HCl salt. The analyticaldata was consistent with that described for the antipode compound 75.

Example 110 Synthesis of Compound 110

Compound 110 was synthesized in a similar manner to that described forcompound 78 and then converted to its hydrochloride salt. ¹HNMR (600MHz, DMSO-d₆) δ ppm 1.50-1.65 (m, 1H) 1.92-2.02 (m, 3H) 2.06-2.15 (m,1H) 2.78 (d, J=3.81 Hz, 4H) 3.10-3.20 (m, 4H) 3.47-3.51 (m, 2H)3.64-3.71 (m, 1H) 3.76-3.83 (m, 2H) 3.98-4.14 (m, 1H) 7.20 (s, 2H) 7.77(s, 1H) 7.97 (s, 2H) 8.81 (s, 1H) 9.03 (s, 1H) 10.97 (br s., 1H). LCMS(ESI) 419 (M+H).

Example 111 Synthesis of Compound 111

Compound 111 was synthesized in a similar manner to that described forcompound 78 and then converted to its hydrochloride salt. ¹HNMR (600MHz, DMSO-d₆) δ ppm 1.54-1.59 (m, 1H) 1.92-2.01 (m, 3H) 2.06-2.15 (m,1H) 2.76-2.84 (m, 1H) 3.17-3.24 (m, 6H) 3.64-3.71 (m, 2H) 4.02-4.11 (m,2H) 7.22 (s, 2H) 7.64 (s, 1H) 7.97 (s, 2H) 8.75 (s, 1H) 8.97 (s, 1H)9.21 (s, 1H). LCMS (ESI) 405 (M+H).

Example 112 Synthesis of Compound 112

Compound 112 was synthesized using similar experimental conditions tothat described for compound 64.

Example 113 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate, Compound113

To a solution of 5-bromo-2,4-dichloropyrimidine (12.80 g, 0.054 mole) inethanol (250 mL) was added Hunig's base (12.0 mL) followed by theaddition of a solution of N-(tert-butoxycarbonyl)-1,2-diaminoethane (10g, 0.0624 mole) in ethanol (80 mL). The contents were stirred overnightfor 20 hrs. The solvent was evaporated under vacuum. Ethyl acetate (800mL) and water (300 mL) were added and the layers separated. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum. Column chromatography on silica gel using hexane/ethyl acetate(0-60%) afforded tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)351 (M+H).

Example 114 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate, Compound 114

To tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate (5 g, 14.23mmole) in toluene (42 mL) and triethylamine (8.33 mL) under nitrogen wasadded triphenyl arsine (4.39 g), 3,3-diethoxyprop-1-yne (3.24 mL) andPddba (1.27 g). The contents were heated at 70 degrees for 24 hrs. Afterfiltration through CELITE®, the crude reaction was columned usinghexane/ethyl acetate (0-20%) to afford the desired product 3.9 g).Column chromatography of the resulting residue using hexane/ethylacetate (0-30%) afforded tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.LCMS (ESI) 399 (M+H).

Example 115 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 115

To a solution of Compound 114 (3.9 g, 0.00976 mole) in THF (60 mL) wasadded TBAF (68.3 mL, 7 eq). The contents were heated to 45 degrees for 2hrs. Concentration followed by column chromatography using ethylacetate/hexane (0-50%) afforded tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale brown liquid (1.1 g). ¹HNMR (d6-DMSO) δ ppm 8.88 (s, 1H), 6.95(brs, 1H), 6.69 (s, 1H), 5.79 (s, 1H), 4.29 (m, 2H), 3.59 (m, 4H), 3.34(m, 1H), 3.18 (m, 1H), 1.19 (m, 9H), 1.17 (m, 6H). LCMS (ESI) 399 (M+H).

Example 116 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 116

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.1 g, 0.00025 mol) in acetonitrile (2 mL) was added1,3-diiodo-5,5-dimethylhydantoin (95 mg, 1 eq), and solid NaHCO₃ (63 mg,3 eq). The reaction was stirred at room temperature for 16 hrs. Thereaction was filtered and concentrated in vacuo. The product waspurified by silica gel column chromatography using hexane/ethylacetate(0-50%) to afford tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale yellow solid (0.03 g). LCMS (ESI) 525 (M+H).

Example 117 Synthesis of tert-ButylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 117

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.1 g, 0.19 mmole) in dioxane (3 mL) was added 2-methylphenylboronicacid (28 mg), tetrakis(triphenylphosphine)palladium (25 mg) andpotassium phosphate (250 mg) in water (0.3 mL). The reaction was heatedin a CEM Discovery microwave at 90° C. for 3 hrs. The crude reaction wasloaded onto silica gel and columned using hexane/ethyl acetate (0-30%)to afford tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.06 g). LCMS (ESI) 489 (M+H).

Example 118 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 118

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.85 g, 1.74 mmole) in AcOH (10 mL) was added water (1.5 mL). Thereaction was stirred at room temperature for 16 hrs. The crude reactionwas then concentrated under vacuum. After the addition of ethyl acetate(50 mL), the organic layer was washed with satd.

NaHCO₃. The organic layer was dried with magnesium sulfate and thenconcentrated under vacuum to afford the crude intermediate, tert-butylN-[2-[2-chloro-6-formyl-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.To this crude intermediate in DMF (5 mL) was added oxone (1.3 g). Afterstirring for 2.5 hrs, water (20 mL) and ethyl acetate (100 mL) wereadded. The organic layer was separated, dried and then concentratedunder vacuum to afford the crude product which was columned over silicagel using hexane/ethyl acetate (0-50%) to afford7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.112 g). LCMS (ESI) 431 (M+H).

Example 119 Synthesis of Compound 119

To7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.1 g, 0.261 mmol) in DCM (4.1 mL) was added DMAP (20 mg) followedby the addition of N,N′-diisopropylcarbodiimide (0.081 mL, 2 eq). Afterstirring for 3 hrs, TFA (0.723 mL) was added. Stirring was thencontinued for another 30 minutes. The reaction mixture was neutralizedwith satd. NaHCO₃. DCM (20 mL) was then added and the organic layerseparated, dried with magnesium sulfate and then concentrated undervacuum to afford the crude product which was columned usinghexane/ethylacetate (0-100%) to afford chloro tricyclic amide Compound119 (0.65 g). LCMS (ESI) 313 (M+H).

Example 120 Synthesis of Compound 120

To the chloro tricyclic amide (0.040 g, 0.128 mmole) (Compound 119) indioxane (2.5 mL) under nitrogen was added Pd₂(dba)₃ (12 mg), sodiumtert-butoxide (16 mg), BINAP (16 mg) and 4-morpholinoaniline (22.7 mg, 1eq). The reaction mixture was heated at 90° C. in a CEM Discoverymicrowave for 3.0 hrs. The crude reaction was loaded onto a silica gelcolumn and the contents eluted with DCM/MeOH (0-6%) to afford theproduct (10 mg). LCMS (ESI) 455 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δ ppm2.14 (s, 3H) 3.23-3.50 (m, 2H) 3.57-3.73 (m, 2H), 3.81-3.92 (m, 8H),7.11-7.31 (m, 4H) 7.31-7.48 (m, 1H) 7.58-7.73 (m, 1H) 7.77-7.95 (m, 2H)8.05-8.21 (m, 1H) 8.44 (s, 1H) 9.85-10.01 (m, 1H).

Example 121 Synthesis of Compound 121

To the chloro tricyclic amide (0.024 g) (Compound 119) inN-methyl-2-pyrrolidone (NMP) (1.5 mL) was addedtrans-4-aminocyclohexanol (0.0768 mmol, 26.54 mg, 3 eq) and Hunig's base(0.4 mL). The reaction was heated in a CEM Discovery microwave vessel at150° C. for 1.2 hrs. The crude reaction was loaded onto a silica gelcolumn and the contents eluted with DCM/MeOH (0-10%) to afford theproduct (21 mg). LCMS (ESI) 392 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.23 (d, J=8.78 Hz, 4H) 1.84 (br. s., 4H) 2.11 (s, 3H) 3.34-3.43 (m, 1H)3.55 (br. s., 2H) 3.72 (br. s., 1H) 4.13 (br. s., 2H) 4.50 (br. s., 1H)7.03 (br. s., 1H) 7.12-7.28 (m, 4H) 7.96 (br. s., 1H) 8.18 (br. s., 1H).

Example 122 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 122

7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 341 (M+H).

Example 123 Synthesis of Compound 123

Chloro tricyclic amide, Compound 123, was synthesized using a similarexperimental procedure as that described for the synthesis of chlorotricyclic amide (Compound 119). LCMS (ESI) 223 (M+H).

Example 124 Synthesis of Compound 124

To the chloro tricyclic amide, Compound 123 (0.035 g, 0.00157 mole) inNMP (1.5 mL) was added Hunig's base (0.3 mL) followed by the addition ofthe trans-4-aminocyclohexanol (54.2 mg). The reaction mixture was heatedat 150° C. for 1.5 hrs. The crude reaction was loaded onto a silica gelcolumn and the column was eluted with DCM/MeOH (0-10%) to afford theproduct (5 mg). LCMS (ESI) 302 (M+H).

Example 125 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamate,Compound 125

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-(2-amino-2-methyl-propyl)carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)(M+H) 379.

Example 126 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate,Compound 126

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewas synthesized by treating tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate. 10 LCMS (ESI) (M+H) 427.

Example 127 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-2-methyl-propyl]carbamate,Compound 127

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-2-methyl-propyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.LCMS (ESI) (M+H) 427.

Example 128 Synthesis of7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 128

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 369 (M+H).

Example 129 Synthesis of Compound 129

Chloro tricyclic amide, Compound 129, was synthesized using a similarprocedure as that described for the synthesis of chloro tricyclic amide,Compound 119. LCMS (ESI) 251 (M+H).

Example 130 Synthesis of Compound 130

Compound 130 was synthesized by treating chlorotricyclic amine Compound129 with trans-4-aminocyclohexanol using similar experimental conditionsas for compound 124. LCMS (ESI) 330 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δppm 1.07-1.34 (m, 4H) 1.47-2.05 (m, 10H) 3.09 (m, 1H) 3.51 (d, J=2.91Hz, 2H) 3.57 (m, 1H) 4.50 (br. s., 1H) 6.89 (s, 1H) 6.94-7.05 (m, 1H)8.04 (br. s., 1H) 8.60 (s, 1H) 9.00 (br. s., 1H).

Example 131 Synthesis of benzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]carbamate,Compound 131

BenzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine with benzylN-[1-(aminomethyl)propyl]carbamate using similar experimental conditionsas described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)(M+H) 413.

Example 132 Synthesis of benzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamate,Compound 132

BenzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamatewas prepared by treating benzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]-carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamateLCMS (ESI) (M+H) 461.

Example 133 Synthesis of benzylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]propyl]carbamate,Compound 133

BenzylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]propyl]carbamatewas synthesized by treating benzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) (M+H) 461.

Example 134 Synthesis of7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 134

7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 403 (M+H).

Example 135 Synthesis of Compound 135

To a solution of7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid in dichloromethane was added HBr, the reaction was stirred at 45degrees for 3 hrs. After concentration, 2N NaOH was added to basify(pH=8.0) the reaction followed by the addition of THF (20 mL). Boc₂O wasthen added (1.2 eq) and the reaction was stirred for 16 hrs. To thecrude reaction mixture was then added ethyl acetate (100 mL) and water(50 mL) and the organic phase was separated, dried (magnesium sulfate)and then concentrated under vacuum. To the crude product was addeddichloromethane (30 mL) followed by DIC and DMAP. After stirring for 2hrs, TFA was added and the contents stirred for an hour. The solventswere evaporated under vacuum and the residue basified with satd. NaHCO₃.Ethyl acetate was then added and the organic layer separated, dried(magnesium sulfate) and then concentrated under vacuum. Columchromatography with hexane/ethyl acetate (0-100%) afforded the desiredchlorotricyclic core, Compound 135. LCMS (ESI) 251 (M+H).

Example 136 Synthesis of Compound 136

Compound 136 was synthesized by treating chlorotricyclic amine, Compound135, with trans-4-aminocyclohexanol using similar experimentalconditions as for compound 124. LCMS (ESI) 330 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.80-0.95 (m, 3H) 1.35-1.92 (m, 10H) 3.66 (br. m., 3H)4.17 (br. s., 2H) 4.47 (br. s., 1H) 6.85 (s, 1H) 6.96 (br. s., 1H) 8.15(br. s., 1H) 8.62 (br. s., 1H).

Example 137 Synthesis of tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamate,Compound 137

tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-[1-(aminomethyl)cyclopentyl]carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)405 (M+H).

Example 138 Synthesis of tert-butylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]cyclopentyl]carbamate,Compound 138

tert-butylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate LCMS (ESI) 453 (M+H).

Example 139 Synthesis of tert-butylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]cyclopentyl]carbamate,Compound 139

tert-butylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) 453 (M+H).

Example 140 Synthesis of7-[[1-(tert-butoxycarbonylamino)cyclopentyl]methyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 140

7-[[1-(tert-butoxycarbonylamino)cyclopentyl]methyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 395 (M+H).

Example 141 Synthesis of Compound 141

Chlorotricyclic core Compound 141 was synthesized using a similarexperimental procedure as that described for the synthesis of chlorotricyclic amide Compound 119. LCMS (ESI) 277 (M+H).

Example 142 Synthesis of Compound 142

Compound 142 was synthesized by treating chlorotricyclic amine, Compound141, with trans-4-aminocyclohexanol using similar experimentalconditions as for Compound 124. LCMS (ESI) 356 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.08-1.32 (m, 8H) 1.60-2.09 (m, 8H) 3.03-3.17 (m, 1H)3.35 (s, 2H) 3.54-3.62 (m, 1H) 4.51 (d, J=4.39 Hz, 1H) 6.88 (s, 1H) 6.96(br. s., 1H) 8.07 (br. s., 1H) 8.58 (s, 1H).

Example 143 Synthesis of tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate,Compound 143

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-[(1-aminocyclopentyl)methyl]carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)405 (M+H).

Example 144 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate,Compound 144

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamatewas synthesized by treating tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate.

LCMS (ESI) 453 (M+H).

Example 145 Synthesis of tert-butylN-[[1-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentyl]methyl]carbamate,Compound 145

tert-ButylN-[[1-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentyl]methyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) 4534 (M+H).

Example 146 Synthesis of7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6carboxylicacid, Compound 146

7-[2-(tert-Butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 395 (M+H).

Example 147 Synthesis of Compound 147

Chloro tricyclic amide, Compound 147 was synthesized using a similarexperimental procedure as that described for the chloro tricyclic amide,Compound 119. LCMS (ESI) 277 (M+H).

Example 148 Synthesis of Compound 148

Compound 148 was synthesized by treating chlorotricyclic amine, Compound147, with trans-4-aminocyclohexanol using similar experimentalconditions as for Compound 124. LCMS (ESI) 356 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.06-1.35 (m, 8H) 1.45-1.95 (m, 8H) 3.10 (m, 1H) 3.58(br. s., 2H) 3.95 (br. s., 1H) 4.49 (br. s., 1H) 6.84 (s, 1H) 6.85-6.93(m, 1H) 8.29 (s, 1H) 8.61 (br. s., 1H).

Example 149 Synthesis of Compound 149

-   -   Step 1: Compound 59 is Boc protected according to the method        of A. Sarkar et al. (JOC, 2011, 76, 7132-7140).    -   Step 2: Boc-protected Compound 59 is treated with 5 mol %        NiCl₂(Ph₃)₂, 0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq        tetraethylammonium iodide, in DMI under CO₂ (1 atm) at 25° C.        for 20 hours to convert the aryl halide derivative into the        carboxylic acid.    -   Step 3: The carboxylic acid from Step 2 is converted to the        corresponding acid chloride using standard conditions.    -   Step 4: The acid chloride from Step 3 is reacted with N-methyl        piperazine to generate the corresponding amide.    -   Step 5: The amide from Step 4 is deprotected using        trifluoroacetic acid in methylene chloride to generate the        target compound. Compound 149 is purified by silica gel column        chromatography eluting with a dichloromethane-methanol gradient        to provide Compound 149.

Each of Compounds 119 through 149 and corresponding compounds withvarious R⁸, R¹ and Z definitions may be reacted with sodium hydride andan alkyl halide or other halide to insert the desired R substitutionprior to reaction with an amine, such as described above for thesynthesis of Compound 120, to produce the desired product of Formulae I,II, III, IV, or V.

Example 150 CDK4/6 Inhibition In Vitro Assay

Selected compounds disclosed herein were tested in CDK4/cyclinD1,CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (SantaClara, CA) to determine their inhibitory effect on these CDKs. Theassays were performed using microfluidic kinase detection technology(Caliper Assay Platform). The compounds were tested in 12-pointdose-response format in singlicate at Km for ATP. Phosphoacceptorsubstrate peptide concentration used was 1 M for all assays andStaurosporine was used as the reference compound for all assays.Specifics of each assay are as described below:

-   -   CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration:        50 μM; Incubation time: 3 hr.    -   CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration:        100 μM; Incubation time: 1 hr.    -   CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration:        200 μM; Incubation time: 10 hr.    -   CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration:        300 μM; Incubation time: 3 hr.

The inhibitory IC₅₀ values for the compounds in Table 1 for CDK4/CycD1,CDK2/CycE, CDK2/CycA, as well as fold selectivity are presented in Table2.

TABLE 2 Selective Inhibition of CDK4 CDK4/ CDK2/ Fold CDK2/ Fold CycD1CycE Selectivity CycA Selectivity IC₅₀ IC₅₀ (CDK2/CycE/ IC₅₀ (CDK2/CycA/Structure [nM] [nM] CDK4) [nM] CDK4) A 4.2 6350 1516 3160 754 B 0.4 30406862 1890 4266 C 1.4 1920 1333 616 428 D 0.9 3480 3779 1500 1629 E 1 695688 204 202 F 1.5 628 419 190 127 G 1.5 2580 1767 646 442 H 1.5 15201013 377 251 I 2 2120 1065 1130 568 J 0.7 5110 7707 4340 6546 K 1 10701019 738 703 L 5.7 4530 789 1490 260 M 2.3 2280 1004 1410 621 N 1 15001500 ND ND O 2.5 41410 1636 3150 1245 P 3.3 3560 1085 1010 308 Q 0.61080 1722 3030 4833 R 0.5 1920 3918 1360 2776 S 1.7 1250 718 342 197 T0.8 1660 2022 1670 2034 U 0.7 1460 2229 857 1308 V 2.9 3500 1224 2130745 W 2.7 3970 1481 539 201 X 0.9 11600 12975 1840 2058 Y 2.5 124 50 6125 Z 3.2 3710 1174 647 205 AA 0.5 6100 13319 4630 10109 BB 0.8 1680 2017502 603 CC 1.6 1250 791 755 478 DD 1.9 9620 5200 8360 4519 EE 3.8 1660432 1110 289 FF 1.2 4620 3949 1400 1197 GG 1 3580 3377 1510 1425 HH 1.71280 766 265 159 II 2 367 184 239 120 JJ 1.4 288 204 ND ND KK 2.3 1760762 915 396 LL 2 202 103 108 55 MM 1.8 3390 1863 597 328 NN 3.7 47001274 1560 423 OO 9 3980 442 570 63 PP 3.1 3600 1146 3090 984 QQ 4.1 3060746 2570 627 RR 1.2 1580 1374 693 603 SS 0.8 1460 1865 1390 1775 TT 0.81260 1550 596 733 UU 7.3 3960 542 ND ND VV 3.3 2630 809 789 243 WW 0.71350 204 ND ND XX 1.3 7300 5615 6290 4838 YY 4.6 6900 1490 ND ND ZZ 10.59960 949 ND ND AAA 2.3 6010 2591 2130 918 BBB 2.8 187 68 85 31 CCC 22170 1074 457 226 DDD 9.5 9350 986 ND ND EEE 2.3 2950 1266 943 405 FFF4.7 4540 966 1370 291 GGG 13.7 7610 555 ND ND HHH 6.8 2840 419 ND ND III6 3770 626 ND ND JJJ 3.2 5200 1620 2830 882 KKK 1.3 291 231 87.3 69 LLL3.2 1620 509 4530 1425 MMM 3.2 1890 600 990 314 NNN 1.4 2930 2154 1010743 OOO 2.4 393 164 203 85 PPP 0.8 16500 21263 2640 3402 QQQ 10.5 111001057 ND ND RRR 2.6 4500 1758 ND ND SSS 2 2280 1112 1880 917 TTT 3.4 3030899 ND ND UUU 18 16460 914 ND ND VVV 7.4 4380 589 ND ND WWW 18.5 2500135 ND ND XXX 11.4 6620 581 ND ND

To further characterize its kinase activity, Compound T was screenedagainst 456 (395 non-mutant) kinases using DiscoveRx's KINOMEscan™profiling service. The compound was screened using a singleconcentration of 1000 nM (>1000 times the IC50 on Cdk4). Results fromthis screen confirmed the high potency against Cdk4 and high selectivityversus Cdk2. Additionally, the kinome profiling showed that Compound Twas relatively selective for Cdk4 and Cdk6 compared to the other kinasestested. Specifically, when using an inhibitory threshold of 65%, 90%, or99%, Compound T inhibited 92 (23.3%), 31 (7.8%) or 6 (1.5%) of 395non-mutant kinases respectively.

In addition to CDK4 kinase activity, several compounds were also testedagainst CDK6 kinase activity. The results of the CDK6/CycD3 kinaseassays, along with the CDK4/cyclinD1, CDK2/CycA and CDK2/cyclinE kinaseassays, are shown for PD0332991 (Reference) and the compounds T, Q, GG,and U in Table 3. The IC₅₀ of 10 nM for CDK4/cyclinD1 and 10 uM forCDK12/CyclinE agrees well with previously published reports forPD0332991 (Fry et al. Molecular Cancer Therapeutics (2004)3(11)1427-1437; Toogood et al. Journal of Medicinal Chemistry (2005) 48,2388-2406). Compounds T, Q, GG, and U are more potent (lower IC₅₀) withrespect to the reference compound (PD0332991) and demonstrate a higherfold selectivity with respect to the reference compound (CDK2/CycE IC₅₀divided by CDK4/CycD1 IC₅₀).

TABLE 3 Inhibition of CDK kinases by Compounds T, Q, GG, and U CDK4/CDK2/ Fold CycD1 CycE Selectivity CDK2/ CDK6/ IC₅₀ IC₅₀ CDK2/ CycA CycD3Formula (nM) (uM) CDK4 IC₅₀(uM) IC₅₀(nM) PD0332991 10 10 1000 Not NotReference determined determined Compound T 0.821 1.66 2022 1.67 5.64Compound Q 0.627 1.08 1722 3.03 4.38 Compound GG 1.060 3.58 3377 1.514.70 Compound U 0.655 1.46 2229 .857 5.99

Example 151 G1 Arrest (Cellular G1 and S-phase) Assay

For determination of cellular fractions in various stages of the cellcycle following various treatments, HS68 cells (human skin fibroblastcell line (Rb-positive)) were stained with propidium iodide stainingsolution and run on Dako Cyan Flow Cytometer. The fraction of cells inG0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle wasdetermined using FlowJo 7.2.2 analysis.

The compounds listed in Table 1 were tested for their ability to arrestHS68 cells at the G1 phase of the cell cycle. From the results of thecellular G1 arrest assay, the range of the inhibitory EC₅₀ valuesnecessary for G1 arrest of HS68 cells was from 22 nM to 1500 nM (seecolumn titled “Cellular G1 Arrest EC₅₀” in Table 4).

Example 152 Cell Cycle Arrest by Compound T in Cdk4/6-Dependent Cells

To test the ability of Cdk4/6 inhibitors to induce a clean G1-arrest, acell based screening method was used consisting of two Cdk4/6-dependentcell lines (tHS68 and WM2664; Rb-positive) and one Cdk4/6-independent(A2058; Rb-negative) cell line. Twenty-four hours after plating, eachcell line was treated with Compound T in a dose dependent manner for 24hours. At the conclusion of the experiment, cells were harvested, fixed,and stained with propidium iodide (a DNA intercalator), which fluorescesstrongly red (emission maximum 637 nm) when excited by 488 nm light.Samples were run on Dako Cyan flow cytometer and >10,000 events werecollected for each sample. Data were analyzed using FlowJo 2.2 softwaredeveloped by TreeStar, Inc.

FIG. 2B is a graph of the number of tHS68 cells (CDK4/6-dependent cellline) vs. the DNA content of the cells (as measured by propidiumiodide). Cells were treated with DMSO for 24 hours, harvested, andanalyzed for cell cycle distribution. FIG. 2C is a graph of the numberof WM2664 cells (CDK4/6-dependent cell line) vs. the DNA content of thecells (as measured by propidium iodide). Cells were treated with DMSOfor 24 hours, harvested, and analyzed for cell cycle distribution. FIG.2D is a graph of the number of A2058 cells (CDK4/6-independent cellline) vs. the DNA content of the cells (as measured by propidiumiodide). Cells were treated with DMSO for 24 hours, harvested, andanalyzed for cell cycle distribution. FIG. 2E is a graph of the numberof tHS68 cells (CDK4/6-dependent cell line) vs. the DNA content of thecells (as measured by propidium iodide) after treatment with Compound T.Cells were treated with Compound T (300 nM) for 24 hours, harvested, andanalyzed for cell cycle distribution. As described in Example 152,treatment of tHS68 cells with Compound T causes a loss of the S-phasepeak (indicated by arrow). FIG. 2F is a graph of the number of WM2664cells (CDK4/6-dependent cell line) vs. the DNA content of the cells (asmeasured by propidium iodide) after treatment with Compound T. Cellswere treated with Compound T (300 nM) for 24 hours, harvested, andanalyzed for cell cycle distribution. As described in Example 152,treatment of WM2664 cells with Compound T causes a loss of the S-phasepeak (indicated by arrow). FIG. 2G is a graph of the number of A2058cells (CDK4/6-independent cell line) vs. the DNA content of the cells(as measured by propidium iodide) after treatment with Compound T. Cellswere treated with Compound T (300 nM) for 24 hours, harvested, andanalyzed for cell cycle distribution. As described in Example 152,treatment of A2058 cells with Compound T does not cause a loss of theS-phase peak (indicated by arrow).

Example 153 Compound T Inhibits Phosphorylation of RB

The Cdk4/6-cyclin D complex is essential for progression from G1 to theS-phase of the DNA cell cycle. This complex phosphorylates theretinoblastoma tumor suppressor protein (Rb). To demonstrate the impactof Cdk4/6 inhibition on Rb phosphorylation (pRb), Compound T was exposedto three cell lines, two Cdk4/6 dependent (tHS68, WM2664; Rb-positive)and one Cdk4/6 independent (A2058; Rb-negative). Twenty four hours afterseeding, cells were treated with Compound T at 300 nM finalconcentration for 4, 8, 16, and 24 hours. Samples were lysed and proteinwas assayed by western blot analysis. Rb phosphorylation was measured attwo sites targeted by the Cdk4/6-cyclin D complex, Ser780 and Ser807/811using species specific antibodies. Results demonstrate that Compound Tblocks Rb phosphorylation in Rb-dependent cell lines by 16 hours postexposure, while having no effect on Rb-independent cells (FIG. 3 ).

Example 154 Small Cell Lung Cancer (SCLC) Cells are Resistant to CDK4/6Inhibitors

The retinoblastoma (RB) tumor suppressor is a major negative cell cycleregulator that is inactivated in approximately 11% of all human cancers.Functional loss of RB is an obligate event in small cell lung cancer(SCLC) development. In RB competent tumors, activated Cdk2/4/6 promoteG1 to S phase traversal by phosphorylating and inactivating RB (andrelated family members). Conversely, cancers with RB deletion orinactivation do not require Cdk4/6 activity for cell cycle progression.Since inactivation of RB is an obligate event in SCLC development, thistumor type is highly resistant to Cdk4/6 inhibitors andco-administration of Cdk4/6 inhibitors with DNA damagingchemotherapeutic agents such as those used in SCLC should not antagonizethe efficacy of such agents.

Several compounds (PD0332991, Compound GG, and Compound T) were testedfor their ability to block cell proliferation in a panel of SCLC celllines with known genetic loss of RB. SCLC cells were treated with DMSOor the indicated Cdk4/6 inhibitor for 24 hours. The effect of Cdk4/6inhibition on proliferation was measured by EdU incorporation. AnRB-intact, Cdk4/6-dependent cell line (WM2664 or tHS68) and a panel ofRB-negative SCLC cell lines (H69, H82, H209, H345, NCI417, or SHP-77)were analyzed for growth inhibition by the various CDK4/6 inhibitors.

As shown in FIG. 4 , Rb-negative SCLC cells are resistant to Cdk4/6inhibition. In FIG. 4A, PD0332991 inhibits the Rb-positive cell line(WM2664), but does not affect the growth of the Rb-negative small celllung cancer cell lines (H345, H69, H209, SHP-77, NCI417, and H82). InFIG. 4B, Compound GG inhibits the Rb-positive cell line (tHS68), butdoes not affect the growth of the Rb-negative cell lines (H345, H69,SHP-77, and H82). In FIG. 4C, Compound T inhibits the Rb-positive cellline (tHS68), but does not affect the growth of the Rb-negative celllines (H69, SHP-77, and H209). This analysis demonstrated that RB-nullSCLC cell lines were resistant to Cdk4/6 inhibition, as no change in thepercent of cells in S-phase were seen upon treatment with any of theCdk4/6 inhibitors tested, including Compound T and Compound GG, whilethe RB-proficient cell line in each experiment was highly sensitive toCdk4/6 inhibition with almost no cells remaining in S-phase after 24hours of treatment.

Example 155 Rb-Negative Cancer Cells are Resistant to CDK4/6 Inhibitors

Cellular proliferation assays were conducted using the followingRb-negative cancer cell lines: H69 (human small cell lungcancer—Rb-negative) cells or A2058 (human metastatic melanomacells—Rb-negative). These cells were seeded in Costar (Tewksbury,Massachusetts) 3093 96 well tissue culture treated white walled/clearbottom plates. Cells were treated with the compounds of Table 1 as ninepoint dose response dilution series from 10 uM to 1 nM. Cells wereexposed to compounds and then cell viability was determined after eitherfour (H69) or six (A2058) days as indicated using the CellTiter-Glo®luminescent cell viability assay (CTG; Promega, Madison, Wisconsin,United States of America) following the manufacturer's recommendations.Plates were read on BioTek (Winooski, Vermont) Syngergy2 multi-modeplate reader. The Relative Light Units (RLU) were plotted as a result ofvariable molar concentration and data was analyzed using Graphpad(LaJolla, California) Prism 5 statistical software to determine the EC₅₀for each compound.

Select compounds disclosed herein were evaluated against a small celllung cancer cell line (H69) and a human metastatic melanoma cell line(A2058), two Rb-deficient (Rb-negative) cell lines. The results of thesecellular inhibition assays are shown in Table 4. The range of theinhibitory EC₅₀ values necessary for inhibition of H69 small cell lungcancer cells was 2040 nM to >3000 nM. The range of the inhibitory EC₅₀values necessary for inhibition of A2058 malignant melanoma cellproliferation was 1313 nM to >3000 nM. In contrast to the significantinhibition seen on Rb-positive cell lines, it was found that thecompounds tested were not significantly effective at inhibitingproliferation of the small cell lung cancer or melanoma cells.

TABLE 4 Resistance of Rb-Negative Cancer Cells to CDK4/6 InhibitorsCellular G1 A2058 Arrest EC₅₀ H69 Cellular Cellular EC₅₀ Structure (nM)EC₅₀ [nM] [nM] A 110 >3000 ND B 90 ND ND C 95 ND ND D 50 2911 1670 E 752580 1371 F 175 ND ND G 175 ND ND H 85 2040 1313 I 80 2950 1062 J110 >3000 >3000 K 28 >3000 1787 L 65 2161 >3000 M 100 ND ND N 25 >30001444 O 40 >3000 2668 P 30 >3000 >3000 Q 100 >3000 2610 R 70 >3000 2632 S150 >3000 >3000 T 100 >3000 >3000 U 25 >3000 >3000 V 70 >3000 1353 W160 >3000 >3000 X 65 >3000 >3000 Y 350 ND ND Z 110 ND ND AA 70 >3000 NDBB 75 2943 1635 CC 90 >3000 >3000 DD 100 ND ND EE 125 ND ND FF 80 ND NDGG 80 2920 2691 HH 110 ND ND II 40 >3000 >3000 JJ 90 >3000 >3000 KK 222421 1379 LL 125 >3000 >3000 MM 100 >3000 >3000 NN 110 ND ND OO95 >3000 >3000 PP 100 ND ND QQ 120 >3000 >3000 RR 90 2888 1617 SS 802948 1658 TT 75 ND ND UU 300 ND ND VV 200 ND ND WW 400 ND ND XX 225 NDND YY 175 ND ND ZZ 500 ND ND AAA 275 >3000 >3000 BBB 230 >3000 >3000 CCC250 ND ND DDD 350 ND ND EEE 250 >3000 >3000 FFF 650 ND ND GGG 350 ND NDHHH 250 ND ND III 250 ND ND JJJ 240 ND ND KKK 190 ND ND LLL 250 ND NDMMM 200 >3000 >3000 NNN 210 ND ND OOO 200 >3000 >3000 PPP 275 ND ND QQQ500 ND ND RRR 400 ND ND SSS 1500 ND ND TTT 350 ND ND UUU 300 ND ND VVV300 ND ND WWW 300 ND ND XXX 300 ND ND

Example 156 HSPC Growth Suppression Studies

The effect of PD0332991 on HSPCs has been previously demonstrated. FIG.5 shows the EdU incorporation of mice HSPC and myeloid progenitor cellsfollowing a single dose of 150 mg/kg PD0332991 by oral gavage to assessthe temporal effect of transient CDK4/6 inhibition on bone marrow arrestas reported in Roberts et al. Multiple Roles of Cyclin-Dependent Kinase4/6 Inhibitors in Cancer Therapy. JCNI 2012; 104(6):476-487. As can beseen in FIG. 5 , a single oral dose of PD0332991 results in a sustainedreduction in HSPC (LKS+) and myeloid progenitor cells (LKS−) for greaterthan 36 hours. Not until 48 hours post oral dosing do HSPC and myeloidprogenitor cells return to baseline cell division.

Example 157 Bone Marrow Proliferation as Evaluated Using EdUIncorporation and Flow Cytometry Analysis

For HSPC proliferation experiments, young adult female FVB/N mice weretreated with a single dose as indicated of compound T, compound Q,compound GG or PD0332991 by oral gavage. Mice were then sacrificed atthe indicated times (0, 12, 24, 36, or 48 hours following compoundadministration), and bone marrow was harvested (n=3 mice per timepoint), as previously described (Johnson et al. J. Clin. Invest. (2010)120(7), 2528-2536). Four hours before the bone marrow was harvested,mice were treated with 100 μg of EdU by intraperitoneal injection(Invitrogen). Bone marrow mononuclear cells were harvested andimmunophenotyped using previously described methods and percent EdUpositive cells were then determined (Johnson et al. J. Clin. Invest.(2010) 120(7), 2528-2536). In brief, HSPCs were identified by expressionof lineage markers (Lin−), Sca1 (S+), and c-Kit (K+).

Analysis in mice determined that Compound T, Compound Q, and Compound GGdemonstrated dose dependent, transient, and reversible G1-arrest of bonemarrow stem cells (HSPC) (FIG. 6 ). Six mice per group were dosed byoral gavage at 150 mg/kg of Compound T, Compound Q, Compound GG, orvehicle only. Four hours before animals were sacrificed and the bonemarrow was harvested, mice were treated with 100 μg of EdU byintraperitoneal injection. Three mice per group were sacrificed at 12hours and the remaining three animals per group were sacrificed at 24hours. Results are shown in FIG. 6A as the ratio of EdU positive cellsfor treated animals at 12 or 24 hour time points compared to control.Compound T and GG demonstrated a reduction in EdU incorporation at 12hours which was starting to return to normal at 24 hours. Compound Qalso demonstrated some reduction at 12 hours and started to return tobaseline at 24 hours despite the fact that oral bioavailability ofCompound Q is low.

Further experiments were completed with Compound T examining doseresponse and longer periods of compound treatment. Compound T was dosedby oral gavage at 50, 100 or 150 mg/kg and EdU incorporation into bonemarrow was determined at 12 and 24 hours as described above.Alternatively, Compound T was dosed by oral gavage at 150 mg/kg and EdUincorporation into bone marrow was determined at 12, 24, 36 and 48hours. As can be seen in FIGS. 6B and 5C, and similar to the cellularwashout experiments, bone marrow cells, and in particular HSPCs werereturning to normal cell division as determined by EdU incorporation in24 hours following oral gavage at a number of doses. The 150 mg/kg oraldose of Compound T in FIG. 6C can be compared directly to the results ofthe same dose of PD0332991 shown in FIG. 5 where cells were stillnon-dividing (as determined by low EdU incorporation) at 24 and 36hours, only returning to normal values at 48 hours.

Example 158 HSPC Growth Suppression Studies Comparing Compound T andPD0332991

FIG. 7 is a graph of the percentage of EdU positive HSPC cells for micetreated with either PD0332991 (triangles) or compound T (upside downtriangles) v. time after administration (hours) of the compound. Bothcompounds were administered at 150 mg/kg by oral gavage. One hour priorto harvesting bone marrow, EdU was IP injected to label cycling cells.Bone marrow was harvested at 12, 24, 36, and 48 hours after compoundtreatment and the percentage of EdU positive HSPC cells was determinedat each time point.

As seen in FIG. 7 , a single oral dose of PD0332991 results in asustained reduction in HSPCs for greater than 36 hours. In contrast, asingle oral dose of Compound T results in an initial reduction of HSPCproliferation at 12 hours, but proliferation of HSPCs resumes by 24hours after dosage of Compound T.

Example 159 Cellular Wash-Out Experiment

HS68 cells were seeded out at 40,000 cells/well in 60 mm dish on day 1in DMEM containing 10% fetal bovine serum, 100 U/mlpenicillin/streptomycin and 1× Glutamax (Invitrogen) as described(Brookes et al. EMBO J, 21(12)2936-2945 (2002) and Ruas et al. Mol CellBiol, 27(12)4273-4282 (2007)). 24 hrs post seeding, cells are treatedwith compound T, compound Q, compound GG, compound U, PD0332991, or DMSOvehicle alone at 300 nM final concentration of test compounds. On day 3,one set of treated cell samples were harvested in triplicate (0 Hoursample). Remaining cells were washed two times in PBS-CMF and returnedto culture media lacking test compound. Sets of samples were harvestedin triplicate at 24, 40, and 48 hours.

Alternatively, the same experiment was done using normal Renal ProximalTubule Epithelial Cells (Rb-positive) obtained from American TypeCulture Collection (ATCC, Manassas, VA). Cells were grown in anincubator at 37° C. in a humidified atmosphere of 5% CO2 in RenalEpithelial Cell Basal Media (ATCC) supplemented with Renal EpithelialCell Growth Kit (ATCC) in 37° C. humidified incubator.

Upon harvesting cells, samples were stained with propidium iodidestaining solution and samples run on Dako Cyan Flow Cytometer. Thefraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phaseDNA cell cycle was determined using FlowJo 7.2.2 analysis.

FIG. 8 shows cellular wash-out experiments which demonstrate theinhibitor compounds of the present invention have a short, transientG1-arresting effect in different cell types. Compounds T, Q, GG, and Uwere compared to PD0332991 in either human fibroblast cells(Rb-positive) (FIGS. 8A & 8B) or human renal proximal tubule epithelialcells (Rb-positive) (FIGS. 8C & 8D) and the effect on cell cyclefollowing washing out of the compounds was determined at 24, 36, 40, and48 hours.

As shown in FIG. 8 and similar to results in vivo as shown in FIG. 5 ,PD0332991 required greater than 48 hours post wash out for cells toreturn to normal baseline cell division. This is seen in FIG. 8A andFIG. 8B as values equivalent to those for the DMSO control for eitherthe G0-G1 fraction or the S-phase of cell division, respectively, wereobtained. In contrast, HS68 cells treated with compounds of the presentinvention returned to normal baseline cell division in as little as 24hours or 40 hours, distinct from PD0332991 at these same time points.The results using human renal proximal tubule epithelial cells (FIGS. 8C& 8D) also show that PD0332991-treated cells took significantly longerto return to baseline levels of cell division as compared to cellstreated with compounds T, Q, GG, or U.

Example 160 Pharmacokinetic and Pharmacodynamic Properties of CDK4/6Inhibitors

Compounds of the present invention demonstrate good pharmacokinetic andpharmacodynamic properties. Compound T, Q, GG, and U were dosed to miceat 30 mg/kg by oral gavage or 10 mg/kg by intravenous injection. Bloodsamples were taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours postdosing and the plasma concentration of compound T, Q, GG, or U weredetermined by HPLC. Compound T, GG, and U were demonstrated to haveexcellent oral pharmacokinetic and pharmacodynamic properties as shownin Table 5. This includes very high oral bioavailability (F(%)) of 52%to 80% and a plasma half-life of 3 to 5 hours following oraladministration. Compound T, Q, GG, and U were demonstrated to haveexcellent pharmacokinetic and pharmacodynamic properties when deliveredby intravenous administration. Representative IV and oral PK curves forall four compounds are shown in FIG. 9 .

TABLE 5 Pharmacokinetic and pharmacodynamic properties of CDK4/6inhibitors Compound Compound Compound Compound Mouse PK T Q GG U CL(mL/min/kg) 35 44 82 52 Vss (L/kg) 2.7 5.2 7.5 3.4 t_(1/2) (h) p.o. 50.8 3.5 3 AUC _(0-inf) (uM*h) i.v. 1.3 0.95 1.1 0.76 AUC (uM*h) p.o. 2.90.15 1.9 3.3 C_(max) (uM) p.o. 2.5 0.16 1.9 4.2 T_(max) (h) p.o. 1 0.5 10.5 F (%) 80 2 52 67

Example 161 Metabolic Stability

The metabolic stability of Compound T in comparison to PD0332991 wasdetermined in human, dog, rat, monkey, and mouse liver microsomes.Human, mouse, and dog liver microsomes were purchased from Xenotech, andSprague-Dawley rat liver microsomes were prepared by Absorption Systems.The reaction mixture comprising 0.5 mg/mL of liver microsomes, 100 mM ofpotassium phosphate, pH 7.4, 5 mM of magnesium chloride, and 1 uM oftest compound was prepared. The test compound was added into thereaction mixture at a final concentration of 1 uM. An aliquot of thereaction mixture (without cofactor) was incubated in shaking water bathat 37 deg. C for 3 minutes. The control compound, testosterone, was runsimultaneously with the test compound in a separate reaction. Thereaction was initiated by the addition of cofactor (NADPH), and themixture was then incubated in a shaking water bath at 37 deg. C.Aliquots (100 μL) were withdrawn at 0, 10, 20, 30, and 60 minutes forthe test compound and 0, 10, 30, and 60 minutes for testosterone. Testcompound samples were immediately combined with 100 μL of ice-coldacetonitrile containing internal standard to terminate the reaction.Testosterone samples were immediately combined with 800 μL of ice cold50/50 acetonitrile/dH2O containing 0.1% formic acid and internalstandard to terminate the reaction. The samples were assayed using avalidated LC-MS/MS method. Test compound samples were analyzed using theOrbitrap high resolution mass spectrometer to quantify the disappearanceof parent test compound and detect the appearance of metabolites. Thepeak area response ration (PARR) to internal standard was compared tothe PARR at time 0 to determine the percent of test compound or positivecontrol remaining at time-point. Half-lives were calculated usingGraphPad software, fitting to a single-phase exponential decay equation.

Half-life was calculated based on t½=0.693 k, where k is the eliminationrate constant based on the slope plot of natural logarithm percentremaining versus incubation time. When calculated half-life was longerthan the duration of the experiment, the half-life was expressed as >thelongest incubation time. The calculated half-life is also listed inparentheses. If the calculated half-life is >2× the duration of theexperiment, no half-life was reported. The timely resumption of cellularproliferation is necessary for tissue repair, and therefore an overlylong period of arrest is undesirable in healthy cells such as HSPCs. Thecharacteristics of a CDK4/6 inhibitor that dictate its arrestingduration are its pharmacokinetic (PK) and enzymatic half-lives. Onceinitiated, a G1-arrest in vivo will be maintained as long as circulatingcompound remains at an inhibitory level, and as long as the compoundengages the enzyme. PD032991, for example, possesses an overall long PKhalf-life and a fairly slow enzymatic off-rate. In humans, PD0332991exhibits a PK half-life of 27 hours (see Schwartz, G K et al. (2011)BJC, 104:1862-1868). In humans, a single administration of PD0332991produces a cell cycle arrest of HSPC lasting approximately one week.This reflects the 6 days to clear the compound (5 half-lives×27 hourhalf-life), as well as an additional 1.5 to 2 days of inhibition ofenzymatic CDK4/6 function. This calculation suggests that it takes atotal of 7+ days for normal bone marrow function to return, during whichtime new blood production is reduced. These observations may explain thesevere granulocytopenia seen with PD0332991 in the clinic.

Further experiments were completed with Compound T and PD0332991 tocompare the metabolic stability (half-life) in human, dog, rat, monkey,and mouse liver microsomes. As shown in FIG. 10 , when analyzing thestability of the compounds in liver microsomes across species, thedeterminable half-life of Compound T is shorter in each species comparedto that reported for PD0332991. Furthermore, as previously describedabove and in FIG. 8 , it appears that PD0332991 also has an extendedenzymatic half-life, as evidenced by the production of a pronounced cellcycle arrest in human cells lasting more than forty hours even aftercompound is removed from the cell culture media (i.e., in an in vitrowash-out experiment). As further shown in FIG. 8 , removal of thecompounds described herein from the culture media leads to a rapidresumption of proliferation, consistent with a rapid enzymatic off rate.These differences in enzymatic off rates translate into a markeddifference in pharmacodynamic (PD) effect, as shown in FIGS. 5, 6C, and7 . As shown, a single oral dose of PD0332991 produces a 36+ hour growtharrest of hematopoietic stem and progenitor cells (HSPCs) in murine bonemarrow, which is greater than would be explained by the 6 hour PKhalf-life of PD0332991 in mice. In contrast, the effect of Compound T ismuch shorter, allowing a rapid re-entry into the cell cycle, providingexquisite in vivo control of HSPC proliferation.

Example 162 Compound T Prevents Chemotherapy-Induced Cell Death, DNADamage, and Caspase Activation

In order to demonstrate that pharmacological quiescence induced byCompound T treatment affords resistance to chemotherapeutic agents withdiffering mechanisms of action, an in vitro model was developed usingtelomerized human diploid fibroblasts (tHDFs; a human foreskinfibroblast line immortalized with expression of human telomerase). Thesecells are highly Cdk4/6-dependent for proliferation as demonstrated bytheir complete G1 arrest following treatment with Cdk4/6 inhibitors (SeeRoberts P J, et al. Multiple Roles of Cyclin-Dependent Kinase 4/6Inhibitors in Cancer Therapy. J Natl Cancer Inst 2012; March 21; 104(6):476-87). Cell survival was determined by Cell TiterGlo assay permanufacturer's recommendations. For both γ-H2AX and caspase 3/7 assays,cells were plated and allowed to become adherent for 24 hours. Cellswere then treated with Compound T (at indicated concentrations) orvehicle control for 16 hours, at which time the indicated chemotherapywas added to the pretreated cells. For γ-H2AX, cells were harvested foranalysis 8 hours after chemotherapy exposure. For the γ-H2AX assay,cells were fixed, permeabilized, and stained with anti-γ-H2AX as per theγ-H2AX Flow Kit (Millipore) and quantitated by flow cytometry. Data wasanalyzed using FlowJo 2.2 software developed by TreeStar, Inc. For thein vitro caspase 3/7 assay, cells were harvested 24 hours postchemotherapy treatment. Caspase 3/7 activation was measured using theCaspase-Glo© 3/7 Assay System (Promega) per manufacturer'srecommendations.

As shown in FIG. 11 , Compound T provides selective protection fromcarboplatin and etoposide-induced cell death. Treatment of tHS68 humanfibroblasts with increasing concentrations of Compound T in the presenceof etoposide (5 μM; FIG. 11A) or carboplatin (100 μM; FIG. 111B)selectively induces a dose dependent cell survival as determined by CellTiterGlo.

Treatment with Compound T prior to treatment with several DNA damagingagents (e.g., carboplatin, doxorubicin, etoposide, camptothecin) or ananti-mitotic (paclitaxel) attenuated DNA damage as measured by γ-H2AXformation (FIG. 12A). Additionally, treatment of tHDF cells withCompound T prior to carboplatin, doxorubicin, etoposide, camptothecin,and paclitaxel exposure elicited a robust decrease in caspase 3/7activation in a dose-dependent manner (FIG. 12B). These data show that atransient cell cycle arrest in G1, induced by Cdk4/6 inhibition,decreases the toxicity of a variety of commonly used cytotoxicchemotherapy agents associated with myelosuppression in Cdk4/6-sensitivecells.

Example 163

Compound T inhibits proliferation of hematopoietic stem and/orprogenitor cells (HSPCs) To characterize the effects of Compound Ttreatment on proliferation of the different mouse hematopoietic cells,8-week-old female C57Bl/6 mice were given a single dose of vehicle alone(20% Solutol) or Compound T (150 mg/kg) by oral gavage. Ten-hours later,all mice were given a single i.p. injection of 100 mcg EdU(5-ethynyl-2′-deoxyuridine) to label cells in S-phase of the cell cycle.All treated mice were euthanized 2 hours after EdU injection, bonemarrow cells were harvested and processed for flow cytometric analysisof EdU-incorporation (FIG. 13 ).

In FIG. 13 , representative contour plots show proliferation in WBM(whole bone marrow; top) and HSPCs (hematopoietic stem and progenitorcells; LSK; bottom), as measured by EdU incorporation for cells with notreatment, EdU treatment only, or EdU plus Compound T treatment.Compound T was found to reduce proliferation of whole bone marrow andhematopoietic stem and progenitor cells.

Compared to vehicle-treated mice, Compound T treated mice showedsignificantly less EdU-positive (EdU⁺) cells in all hematopoieticlineages analyzed. The reduction in EdU⁺ cell frequency is most likelydue to reduced S-phase entry, which is consistent with the fact thatCompound T potently inhibits Cdk4/6 activity. Overall, Compound Ttreatment caused ˜70% reduction of EdU⁺ cell frequency in unfractionatedwhole bone marrow cells (See FIG. 13 and FIG. 14 ). In the hematopoieticstem and progenitor cells (HSPC), Compound T treatment resulted inpotent cell cycle arrest of hematopoietic stem cells (HSC, 74%inhibition), the most primitive cells in the entire hematopoieticlineage hierarchy, as well as multipotent progenitors (MPP, 90%inhibition), the immediate downstream progeny of HSCs (FIG. 14A).

As shown in FIG. 14B, further down the lineage differentiationhierarchy, proliferation of the lineage restricted myeloid (CMP, GMP andMEP) and lymphoid progenitors (CLP) were also significantly inhibited byCompound T, showing between a 76-92% reduction in EdU⁺ cell frequency.

Example 164 Compound T Inhibits Proliferation of DifferentiatedHematopoietic Cells

Using the same experimental protocol as discussed in Example X above andshown in FIGS. 13 and 14 , the effects of Compound T on theproliferation of differentiated hematopoietic cells was investigated.The resulting effect of Compound T in differentiated hematopoietic cellswas more variable than that seen in HSPCs. While T and B cellprogenitors are highly sensitive to Compound T (>99% and >80% reductionin EdU⁺ cell frequencies respectively), proliferation of differentiatedmyeloerythroid cells are more resistant to Compound T, with Mac1G1⁺myeloid cells showing 46% reduction in EdU⁺ cell frequency, and Ter119⁺erythroid cells showing 58% reduction in EdU⁺ cell frequency (FIG. 15 ).Together, these data suggest that while all hematopoietic cells aresensitive to Compound T-induced cell cycle arrest, the degree ofinhibition varies among different cell lineages, with myeloid cellsshowing a smaller effect of Compound T on cell proliferation than seenin the other cell lineages.

Example 165 Compound GG Protects Bone Marrow Progenitors

To assess the effect of transient CDK4/6 inhibition by Compound GG oncarboplatin-induced cytotoxicity in the bone marrow, FVB/n mice (n=3 pergroup) were treated with vehicle control, 90 mg/kg carboplatin byintraperitoneal injection, or 150 mg/kg Compound GG by oral gavage plus90 mg/kg carboplatin by intraperitoneal injection. 24 hours aftertreatment bone marrow was harvested and the percent of cycling bonemarrow progenitors was measured by EdU incorporation as explainedearlier. As shown in FIG. 16 , administration of Compound GG at the sametime as carboplatin administration results in a significant protectionof bone marrow progenitors. EdU incorporation in control animals wasnormalized to 100% and compared to EdU incorporation for bone marrowfrom carboplatin treated animals or carboplatin and Compound GG treatedanimals.

Example 166 Compound T Decreases 5FU-Induced Myelosuppression

To determine the ability of Compound T to modulate chemotherapy-inducedmyelosuppression, a well characterized single-dose 5-fluorouracil (5FU)regimen, known to be highly myelosuppressive in mice, was utilized.FVB/n female mice were given single oral doses of vehicle or Compound Tat 150 mg/kg, followed 30 minutes later by a single intraperitoneal doseof 5FU at 150 mg/kg. Complete blood cell counts were measured every twodays starting on day six.

Co-administration of Compound T positively impacted recovery of allhematopoietic lineages from 5-FU induced myelosuppression. FIG. 17demonstrates the time course of recovery of different blood cell typesin mice treated with Compound T or vehicle control prior to 5FUadministration. It was determined that in each hematopoietic celllineage tested (whole blood cells, neutrophils, lymphocytes, platelets,and red blood cells), Compound T provided a more rapid recovery of thatcells treated only with 5FU. These data show that Compound T treatmentlikely decreases 5FU-induced DNA damage in HSPCs, leading to acceleratedblood count recovery post-chemotherapy.

FIG. 18 shows the data from day 14 of the myelosuppression studiesdescribed above and shown in FIG. 17 . Complete blood cell counts wereanalyzed on day 14. FIG. 18 shows the results for white blood cells(FIG. 18A), neutrophils (FIG. 18B), lymphocytes (FIG. 18C), red bloodcells (FIG. 18D), and platelets (FIG. 18E). In all cases, Compound Twhen administered with 5FU resulted in a significant protection of eachcell type at Day 14 as compared to the myelosuppressive effect of 5FUtreatment alone.

Example 167 Compound T Decreases 5FU-Induced Myelosuppression ThroughRepeated Cycles of 5FU Treatment

To determine the ability of Compound T to modulate chemotherapy-inducedmyelosuppression, a well characterized 5-fluorouracil (5FU) regimen,known to be highly myelosuppressive in mice was utilized. 8-week-oldfemale C57Bl/6 mice were given a single oral dose of vehicle (20%Solutol) or Compound T at 150 mg/kg followed 30 minutes later by anintraperitoneal dose of 5FU at 150 mg/kg. This was repeated every 21days for 3 cycles. Blood samples were taken for hematology analysis onDay 10 of Cycles 1-3.

Co-administration of Compound T reduced the myelosuppression on Day 10of the third cycle (FIG. 19 ), as well as other cycles (data not shown).In accordance with the single-dose study described above, these datashow that Compound T treatment likely decreases 5FU-induced DNA damagein HSPCs, leading to improved hematopoietic blood cell counts.

Example 168 DNA Cell Cycle Analysis in Human Renal Proximal Tubule Cells

To test the ability of Cdk4/6 inhibitors to induce a clean G1-arrest innon-hematopoietic cells, G1 arrest was examined in human renal proximaltubule cells. The cells were treated with Compound T in a dose dependentmanner for 24 hours. At the conclusion of the experiment, cells wereharvested, fixed, and stained with propidium iodide (a DNAintercalator), which fluoresces strongly red (emission maximum 637 nm)when excited by 488 nm light. Samples were run on Dako Cyan flowcytometer. Data were analyzed using FlowJo 2.2 software developed byTreeStar, Inc. Assays were run in triplicate, and error bars were notdetectable. As seen in FIG. 20 , results show that Compound T induces arobust G1 cell cycle arrest in human renal proximal tubule cells, asnearly all cells are found in the G0-G1 phase upon treatment withincreasing amounts of Compound T.

Example 169

Compound T Protects Renal Proximal Tubule Epithelial Cells fromChemotherapy-Induced DNA Damage

The ability of Cdk4/6 inhibitors to protect human renal proximal tubulecells from chemotherapy induced DNA damage was analyzed using etoposideand cisplatin. The cells were treated with Compound T in a dosedependent manner (10 nM, 30 nM, 100 nM, 300 nM, or 1000 nM). At theconclusion of the experiment, cells were harvested, fixed, and stainedwith propidium iodide (a DNA intercalator), which fluoresces stronglyred (emission maximum 637 nm) when excited by 488 nm light. Samples wererun on Dako Cyan flow cytometer. Data were analyzed using FlowJo 2.2software developed by TreeStar, Inc. As seen in FIG. 21 , results showthat Compound T protects renal proximal tubule epithelial cells fromchemotherapy induced DNA damage, as increasing dosages of Compound T incombination with etoposide or cisplatin cause a decrease in thepercentage of S-phase cells, with a corresponding rise in the percentageof cells in the G0-G1 phase.

Example 170 Compound T Prevents Chemotherapy-Induced DNA Damage andCaspase Activation in Human Renal Proximal Tubule Cells

In order to demonstrate that pharmacological quiescence induced byCDK4/6 inhibitor treatment affords resistance to chemotherapeutic agentsin non-hematopoietic cells, the protective effect of Compound T on humanrenal proximal tubule cells was analyzed. Normal renal proximal tubuleepithelial cells were obtained from American Type Culture Collection(ATCC, Manassas, VA). Cells were grown in an incubator at 37° C. in ahumidified atmosphere of 5% CO2 in Renal Epithelial Cell Basal Media(ATCC) supplemented with Renal Epithelial Cell Growth Kit (ATCC) in 37°C. humidified incubator. Cells were treated with either DMSO or 10 nM,30 nM, 100 nM, 300 nM or 1 uM Compound T in either the absence orpresence of 25 uM cisplatin. For the γ-H2AX assay, cells were fixed,permeabilized, and stained with anti-γ-H2AX as per the γ-H2AX Flow Kit(Millipore) and quantitated by flow cytometry. Data was analyzed usingFlowJo 2.2 software developed by TreeStar, Inc. Caspase 3/7 activationwas measured using the Caspase-Glo 3/7 Assay System (Promega, Madison,WI) by following the manufacturer's instructions.

Treatment of renal proximal tubule cells with Compound T in combinationwith cisplatin attenuated DNA damage as measured by γ-H2AX formation(FIG. 22 ). As seen in FIG. 22 , DNA damage caused by cisplatindecreased in a dose-dependent manner after treatment with Compound T.

The ability of Compound T to protect renal proximal tubule epithelialcells against cisplatin induced apoptosis (caspase 3/7 activation) wasalso investigated. As shown in FIG. 23 , Compound T demonstrated adose-dependent reduction in caspase 3/7 activation in these cells. Thisreduction in caspase 3/7 activity was seen at all three levels ofcisplatin tested (25 uM, 50 uM, or 100 uM). These data show that atransient cell cycle arrest in G1, induced by Cdk4/6 inhibition, canprotect renal proximal tubule cells from chemotherapy-induced DNAdamage.

Example 171 Preparation of Drug Product

The active compounds of the present invention can be prepared forintravenous administration using the following procedure. The excipientshydroxypropyl-beta-cyclodextrin and dextrose can be added to 90% of thebatch volume of USP Sterile Water for Injection or Irrigation withstirring; stir until dissolved. The active compound in the hydrochloridesalt form is added and stirred until it is dissolved. The pH is adjustedwith 1N NaOH to pH 4.3+0.1 and 1N HCl can be used to back titrate ifnecessary. USP Sterile Water for Injection or Irrigation can be used tobring the solution to the final batch weight. The pH is next re-checkedto ensure that the pH is pH 4.3+0.1. If the pH is outside of the rangeadd 1N HCl or 1N NaOH as appropriate to bring the pH to 4.3+0.1. Thesolution is next sterile filtered to fill 50 or 100 mL flint glassvials, stopper, and crimped.

This specification has been described with reference to embodiments ofthe invention. The invention has been described with reference toassorted embodiments, which are illustrated by the accompanyingExamples. The invention can, however, be embodied in different forms andshould not be construed as limited to the embodiments set forth herein.Given the teaching herein, one of ordinary skill in the art will be ableto modify the invention for a desired purpose and such variations areconsidered within the scope of the invention.

What is claimed is:
 1. A method of treating a human with triple negativebreast cancer (TNBC) in need thereof comprising: administering to thehuman an effective amount of a selective CDK4/6 inhibitor of structure:

or a pharmaceutically acceptable salt thereof; and, administering to thehuman an effective amount of a chemotherapeutic agent selected from thegroup consisting of carboplatin, cisplatin, gemcitabine, paclitaxel,doxorubicin, cyclophosphamide, and a combination thereof, wherein theCDK4/6 inhibitor is administered 4 hours or less prior to administrationof the chemotherapeutic agent.
 2. The method of claim 1, wherein thechemotherapeutic agent is carboplatin.
 3. The method of claim 1, whereinthe chemotherapeutic agent is cisplatin.
 4. The method of claim 1,wherein the chemotherapeutic agent is paclitaxel.
 5. The method of claim1, wherein the chemotherapeutic agent is doxorubicin.
 6. The method ofclaim 1, wherein the chemotherapeutic agent is cyclophosphamide.
 7. Themethod of claim 1, wherein the chemotherapeutic agent is gemcitabine. 8.A method of reducing chemotherapy-induced myelosuppression in a humanreceiving chemotherapy for the treatment of bladder cancer comprisingadministering to the human an effective amount of at least one DNAdamaging chemotherapeutic agent and an effective amount of a CDK4/6inhibitor of structure:

or a pharmaceutically acceptable salt thereof; wherein the CDK4/6inhibitor is administered 4 hours or less prior to administration of theDNA damaging chemotherapeutic agent.
 9. The method of claim 8, whereinthe CDK4/6 inhibitor is administered 1/2 hour or less prior toadministration of the DNA damaging chemotherapeutic agent.
 10. Themethod of claim 8, wherein the chemotherapeutic agent is administered tothe human on day 1 every 21 days.
 11. The method of claim 8, wherein theDNA damaging chemotherapeutic agent is administered to the human on days1 and 8 every 21 days.
 12. The method of claim 8, wherein the DNAdamaging chemotherapeutic agent is selected from the group consisting ofcisplatin, carboplatin, campothecin, doxorubicin, gemcitabine, andetoposide.
 13. The method of claim 12, wherein the DNA damagingchemotherapeutic agent is cisplatin.
 14. The method of claim 12, whereinthe DNA damaging chemotherapeutic agent is carboplatin.
 15. The methodof claim 12, wherein the DNA damaging chemotherapeutic agent iscampothecin.
 16. The method of claim 12, wherein the chemotherapeuticagent is doxorubicin.
 17. The method of claim 12, wherein thechemotherapeutic agent is gemcitabine.
 18. The method of claim 12,wherein the chemotherapeutic agent is etoposide.